Antibody-drug conjugates and uses thereof

ABSTRACT

The present invention relates to therapeutic immunoconjugates comprising SN-38 attached to an antibody or antigen-binding antibody fragment. The antibody may bind to Trop-2 or CEACAM5 and the immunoconjugate may be administered at a dosage of between 4 mg/kg and 16 mg/kg, preferably 4, 6, 8, 9, 10, 12, or 16 mg/kg. When administered at specified dosages and schedules, the immunoconjugate can reduce solid tumors in size, reduce or eliminate metastases and is effective to treat cancers resistant to standard therapies, such as radiation therapy, chemotherapy or immunotherapy. Surprisingly, the immunoconjugate is effective to treat cancers that are refractory to or relapsed from irinotecan.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/844,772, filed Sep. 3, 2015, which was a continuation-in-part of U.S.patent application Ser. No. 14/204,698 (now issued U.S. Pat. No.9,226,973), filed Mar. 11, 2014, which was a divisional of U.S. patentapplication Ser. No. 13/948,732 (now issued U.S. Pat. No. 9,028,833),filed Jul. 23, 2013, which claimed the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Application Ser. No. 61/736,684, filed Dec. 13,2012, and 61/749,548, filed Jan. 7, 2013. This application claims thebenefit under 35 U.S.C. 119(e) of provisional U.S. Patent ApplicationSer. No. 62/049,631, filed. Sep. 12, 2014, the entire text of eachpriority application incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 2, 2015, isnamed IMM348US1_SL and is 49,315 bytes in size.

FIELD OF THE INVENTION

This invention relates to antibody-drug conjugates (ADCs) comprising oneor more cytotoxic drug moieties conjugated to an antibody orantigen-binding antibody fragment. Preferably, the antibody is ananti-Trop-2 or anti-CEACAM5 antibody, conjugated to SN-38. Morepreferably, a linker such as CL2A may be used to attach the drug to theantibody or antibody fragment. However, other linkers and other knownmethods of conjugating drugs to antibodies may be utilized. Mostpreferably, the antibody or antigen-binding fragment thereof binds to ahuman antigen. The antibody or fragment may be attached to 1-12, 1-6,1-5, 6-8 or 7-8 copies of drug moiety or drug-linker moiety per antibodyor fragment. More preferably, the drug to antibody ratio may varybetween 1.5:1 to 8:1. The ADCs are of use for therapy of cancers, suchas breast, ovarian, cervical, endometrial, lung, prostate, colon,stomach, esophageal, bladder, renal, pancreatic, thyroid, epithelial andhead-and-neck cancer. The ADC may be of particular use for treatment ofcancers that are resistant to one or more standard anti-cancertherapies, such as triple-negative breast cancer, metastatic pancreaticcancer, metastatic gastrointestinal cancer or metastatic colorectalcancer. The ADCs may be used alone or as a combination therapy, alongwith one or more therapeutic modalities selected from the groupconsisting of surgery, radiation therapy, chemotherapy,immunomodulators, cytokines, chemotherapeutic agents, pro-apoptoticagents, anti-angiogenic agents, cytotoxic agents, drugs, toxins,radionuclides, RNAi, siRNA, a second antibody or antibody fragment, andan immunoconjugate. In preferred embodiments, the combination of ADC andother therapeutic modality exhibits a synergistic effect and is moreeffective to induce cancer cell death than either ADC or othertherapeutic modality alone, or the sum of the effects of ADC and othertherapeutic modality administered individually.

RELATED ART

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer in humans, andvirtually no application in other diseases, such as infectious andautoimmune diseases. The toxic agent is most commonly a chemotherapeuticdrug, although particle-emitting radionuclides, or bacterial or planttoxins, have also been conjugated to MAbs, especially for the therapy ofcancer (Sharkey and Goldenberg, C A Cancer J Clin. 2006 July-August;56(4):226-243) and, more recently, with radioimmunoconjugates for thepreclinical therapy of certain infectious diseases (Dadachova andCasadevall, Q J Nucl Med Mol Imaging 2006; 50(3):193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using verywell-defined conjugation chemistries, often at specific sites remotefrom the MAbs' antigen binding regions; (c) MAb-chemotherapeutic drugconjugates can be made more reproducibly and usually with lessimmunogenicity than chemical conjugates involving MAbs and bacterial orplant toxins, and as such are more amenable to commercial developmentand regulatory approval; and (d) the MAb-chemotherapeutic drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates, particularly to the radiation-sensitivebone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY922:1-10 (2000)). CPTs present specific issues in the preparation ofconjugates. One issue is the insolubility of most CPT derivatives inaqueous buffers. Second, CPTs provide specific challenges for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation; and in potent CPT derivatives, suchas SN-38, the active metabolite of the chemotherapeutic CPT-11, andother C-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of a phenolic hydroxyl at the C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Third, thelability under physiological conditions of the δ-lactone moiety of theE-ring of camptothecins results in greatly reduced antitumor potency.Therefore, the conjugation protocol is performed such that it is carriedout at a pH of 7 or lower to avoid the lactone ring opening. However,conjugation of a bifunctional CPT possessing an amine-reactive groupsuch as an active ester would typically require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety preferably is incorporatedin the linker/spacer connecting the CPTs and the antibodies or otherbinding moieties.

A need exists for more effective methods of preparing and administeringantibody-CPT conjugates, such as antibody-SN-38 conjugates. Preferably,the methods comprise optimized dosing and administration schedules thatmaximize efficacy and minimize toxicity of the antibody-CPT conjugatesfor therapeutic use in human patients.

SUMMARY

In various embodiments, the present invention concerns treatment ofcancer with antibody-drug conjugates (ADCs). The ADC may be used aloneor as a combination therapy with one or more other therapeuticmodalities, such as surgery, radiation therapy, chemotherapy,immunomodulators, cytokines, chemotherapeutic agents, pro-apoptoticagents, anti-angiogenic agents, cytotoxic agents, drugs, toxins,radionuclides, RNAi, siRNA, a second antibody or antibody fragment, oran immunoconjugate. In preferred embodiments, the ADC may be of use fortreatment of cancers for which standard therapies are not effective,such as metastatic pancreatic cancer, metastatic colorectal cancer ortriple-negative breast cancer. More preferably, the combination of ADCand other therapeutic modality is more efficacious than either alone, orthe sum of the effects of individual treatments.

In a specific embodiment, an anti-Trop-2 antibody may be a humanized RS7antibody (see, e.g., U.S. Pat. No. 7,238,785, the Figures and Examplessection of which are incorporated herein by reference), comprising thelight chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2(SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavychain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG,SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6). However, as discussedbelow other anti-Trop-2 antibodies are known and may be used in thesubject ADCs. A number of cytotoxic drugs of use for cancer treatmentare well-known in the art and any such known drug may be conjugated tothe antibody of interest. In a more preferred embodiment, the drugconjugated to the antibody is a camptothecin or anthracycline, mostpreferably SN-38 or a pro-drug form of 2-pyrrolinodoxorubicin (2-PDox)(see, e.g., U.S. patent application Ser. Nos. 14/175,089 and 14/204,698,the Figures and Examples section of each incorporated herein byreference).

In another preferred embodiment, therapeutic conjugates comprising ananti-CEACAM5 antibody (e.g., hMN-14, labretuzumab) and/or ananti-CEACAM6 antibody (e.g., hMN-3 or hMN-15) may be used to treat anyof a variety of cancers that express CEACAM5 and/or CEACAM6, asdisclosed in U.S. Pat. Nos. 7,541,440; 7,951,369; 5,874,540; 6,676,924and 8,267,865, the Examples section of each incorporated herein byreference. Solid tumors that may be treated using anti-CEACAM5,anti-CEACAM6, or a combination of the two include but are not limited tobreast, lung, pancreatic, esophageal, medullary thyroid, ovarian, colon,rectum, urinary bladder, mouth and stomach cancers. A majority ofcarcinomas, including gastrointestinal, respiratory, genitourinary andbreast cancers express CEACAM5 and may be treated with the subjectimmunoconjugates. An hMN-14 antibody is a humanized antibody thatcomprises light chain variable region CDR sequences CDR1 (KASQDVGTSVA;SEQ ID NO:114), CDR2 (WTSTRHT; SEQ ID NO:97), and CDR3 (QQYSLYRS; SEQ IDNO:98), and the heavy chain variable region CDR sequences CDR1 (TYWMS;SEQ ID NO:99), CDR2 (EIHPDSSTINYAPSLKD; SEQ ID NO:100) and CDR3(LYFGFPWFAY; SEQ ID NO:101). An hMN-3 antibody is a humanized antibodythat comprises light chain variable region CDR sequences CDR1(RSSQSIVHSNGNTYLE, SEQ ID NO:102), CDR2 (KVSNRFS, SEQ ID NO:103) andCDR3 (FQGSHVPPT, SEQ ID NO:104) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:105), CDR2 (WINTYTGEPTYADDFKG, SEQ ID NO:106) and CDR3(KGWMDFNSSLDY, SEQ ID NO:107). An hMN-15 antibody is a humanizedantibody that comprises light chain variable region CDR sequencesSASSRVSYIH (SEQ ID NO:108); GTSTLAS (SEQ ID NO:109); and QQWSYNPPT (SEQID NO:110); and heavy chain variable region CDR sequences DYYMS (SEQ IDNO:111); FIANKANGHTTDYSPSVKG (SEQ ID NO:112); and DMGIRWNFDV (SEQ IDNO:113).

The antibody moiety may be a monoclonal antibody, an antigen-bindingantibody fragment, a bispecific or other multivalent antibody, or otherantibody-based molecule. The antibody can be of various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprisinghuman IgG1 hinge and constant region sequences. The antibody or fragmentthereof can be a chimeric, a humanized, or a human antibody, as well asvariations thereof, such as half-IgG4 antibodies (referred to as“unibodies”), as described by van der Neut Kolfschoten et al. (Science2007; 317:1554-1557). More preferably, the antibody or fragment thereofmay be designed or selected to comprise human constant region sequencesthat belong to specific allotypes, which may result in reducedimmunogenicity when the ADC is administered to a human subject.Preferred allotypes for administration include a non-G1m1 allotype(nG1m1), such as G1m3, G1m3,1, G1m3,2, or G1m3,1,2. More preferably, theallotype is selected from the group consisting of the nG1m1, G1m3,nG1m1,2 and Km3 allotypes.

The drug to be conjugated to the antibody or antibody fragment may beselected from the group consisting of an anthracycline, a camptothecin,a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, anitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, anitrosourea, a triazene, a folic acid analog, a taxane, a COX-2inhibitor, a pyrimidine analog, a purine analog, an antibiotic, anenzyme inhibitor, an epipodophyllotoxin, a platinum coordinationcomplex, a vinca alkaloid, a substituted urea, a methyl hydrazinederivative, an adrenocortical suppressant, a hormone antagonist, anantimetabolite, an alkylating agent, an antimitotic, an anti-angiogenicagent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shockprotein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, apro-apoptotic agent, and a combination thereof.

Specific drugs of use may be selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin,2-pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox(pro-2-PDox), cyano-morpholino doxorubicin, doxorubicin glucuronide,endostatin, epirubicin glucuronide, erlotinib, estramustine,epidophyllotoxin, erlotinib, entinostat, estrogen receptor bindingagents, etoposide (VP16), etoposide glucuronide, etoposide phosphate,exemestane, fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferaseinhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101,gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,monomethylauristatin F (MMAF), monomethylauristatin D (MMAD),monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib,streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839.

Preferred optimal dosing of the subject ADCs may include a dosage ofbetween 4 mg/kg and 18 mg/kg, preferably given either weekly, twiceweekly or every other week. The optimal dosing schedule may includetreatment cycles of two consecutive weeks of therapy followed by one,two, three or four weeks of rest, or alternating weeks of therapy andrest, or one week of therapy followed by two, three or four weeks ofrest, or three weeks of therapy followed by one, two, three or fourweeks of rest, or four weeks of therapy followed by one, two, three orfour weeks of rest, or five weeks of therapy followed by one, two,three, four or five weeks of rest, or administration once every twoweeks, once every three weeks or once a month. Treatment may be extendedfor any number of cycles, preferably at least 2, at least 4, at least 6,at least 8, at least 10, at least 12, at least 14, or at least 16cycles. Exemplary dosages of use may include 1 mg/kg, 2 mg/kg, 3 mg/kg,4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg. Preferred dosages are4, 6, 8, 9, 10, 12, 14, 16 or 18 mg/kg. More preferred dosages are 6-12,6-8, 7-8, 8-10, 10-12 or 8-12 mg/ml. The person of ordinary skill willrealize that a variety of factors, such as age, general health, specificorgan function or weight, as well as effects of prior therapy onspecific organ systems (e.g., bone marrow) may be considered inselecting an optimal dosage of ADC, and that the dosage and/or frequencyof administration may be increased or decreased during the course oftherapy. The dosage may be repeated as needed, with evidence of tumorshrinkage observed after as few as 4 to 8 doses. The optimized dosagesand schedules of administration disclosed herein show unexpectedsuperior efficacy and reduced toxicity in human subjects, which couldnot have been predicted from animal model studies. Surprisingly, thesuperior efficacy allows treatment of tumors that were previously foundto be resistant to one or more standard anti-cancer therapies. Moresurprisingly, the treatment has been found effective in tumors that werepreviously resistant to camptothecins, such as irinotecan, the parentcompound of SN-38.

The ADCs are of use for therapy of cancers, such as breast, ovarian,cervical, endometrial, lung, prostate, colon, stomach, esophageal,bladder, renal, pancreatic, thyroid, epithelial or head-and-neck cancer.The ADC may be of particular use for treatment of cancers that areresistant, to one or more standard anti-cancer therapies, such as ametastatic colon cancer, triple-negative breast cancer, a HER+, ER+,progesterone+breast cancer, metastatic non-small-cell lung cancer(NSCLC), metastatic pancreatic cancer, metastatic renal cell carcinoma,metastatic gastric cancer, metastatic prostate cancer, or metastaticsmall-cell lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Preclinical in vivo therapy of athymic nude mice, bearing Capan1 human pancreatic carcinoma, with SN-38 conjugates of hRS7(anti-Trop-2), hPAM4 (anti-MUC5ac), hMN-14 (anti-CEACAM5) ornon-specific control hA20 (anti-CD20).

FIG. 2. Preclinical in vivo therapy of athymic nude mice, bearing BxPC3human pancreatic carcinoma, with anti-TROP2-CL2A-SN-38 conjugatescompared to controls.

FIG. 3A. Structure of doxorubicin. “Me” is a methyl group.

FIG. 3B. Structure of 2-pyrrolinodoxorubicin,(2-PDox). “Me” is a methylgroup.

FIG. 3C. Structure of a prodrug form of2-pyrrolinodoxorubicin,(pro-2-PDox). “Me” is a methyl group and “Ac” isan acetyl group.

FIG. 3D. Structure of a maleimide-activated form of pro-2-PDox, forantibody coupling. “Me” is a methyl group and “Ac” is an acetyl group.

FIG. 4. Therapy in nude mice bearing s.c. human tumor xenografts using2.25 mg/kg protein dose (0.064 mg/kg of drug dose) of MAb-pro-2-PDoxconjugates twice weekly×2 weeks in nude mice with Capan-1 humanpancreatic adenocarcinoma xenografts (n=5).

FIG. 5A. Therapy in nude mice bearing s.c. human tumor xenografts using2.25 mg/kg protein dose (0.064 mg/kg of drug dose) of MAb-pro-2-PDoxconjugates twice weekly×2 weeks in nude mice (n=7) with NCI-N87 humangastric carcinoma xenografts.

FIG. 5B. Therapy in nude mice bearing s.c. human tumor xenografts using2.25 mg/kg protein dose (0.064 mg/kg of drug dose) of MAb-pro-2-PDoxconjugates twice weekly×2 weeks in nude mice (n=7) with MDA-MB-468 humanbreast carcinoma xenografts.

FIG. 5C. Therapy in nude mice bearing s.c. human tumor xenografts using2.25 mg/kg protein dose (0.064 mg/kg of drug dose) of MAb-pro-2-PDoxconjugates twice weekly×2 weeks in nude mice (n=7) with BxPC3 humanpancreatic carcinoma xenografts.

FIG. 6A. In vivo efficacy of pro-2-PDox conjugates in nude mice withNCI-N87 human gastric cancer xenografts. Mice were administered a salinecontrol.

FIG. 6B. In vivo efficacy of pro-2-PDox conjugates in nude mice withNCI-N87 human gastric cancer xenografts. Mice were administered 45 μg ofhA20-pro-2-PDox as indicated by arrows.

FIG. 6C. In vivo efficacy of pro-2-PDox conjugates in nude mice withNCI-N87 human gastric cancer xenografts. Mice were administered 45 μg ofhMN-15-pro-2-PDox as indicated by arrows.

FIG. 6D. In vivo efficacy of pro-2-PDox conjugates in nude mice withNCI-N87 human gastric cancer xenografts. Mice were administered 45 μg ofhRS7-pro-2-PDox as indicated by arrows.

FIG. 6E. In vivo efficacy of pro-2-PDox conjugates in nude mice withNCI-N87 human gastric cancer xenografts. Mice were administered 45 μg ofhLL1-pro-2-PDox as indicated by arrows.

FIG. 6F. In vivo efficacy of pro-2-PDox conjugates in nude mice withNCI-N87 human gastric cancer xenografts. Mice were administered 45 μg ofhMN-14-pro-2-PDox as indicated by arrows.

FIG. 7. Effect of different dosing schedules of hRS7-pro-2-PDox onsurvival in nude mice with NCI-N87 human gastric carcinoma xenografts.

FIG. 8A. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered a saline control.

FIG. 8B. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered 45 μg q4d×4 of hRS7-pro-2-PDox.

FIG. 8C. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered 90 μg weekly×2 ofhRS7-pro-2-PDox.

FIG. 8D. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered a single dose of 180 μghRS7-pro-2-PDox.

FIG. 8E. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered 45 μg q4d×4 of hA20-pro-2-PDox.

FIG. 8F. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered 90 μg weekly×2 ofhA20-pro-2-PDox.

FIG. 8G. Dosing schedule study in mice injected with NCI-N87 humangastric cancer. Mice were administered a single dose of 180 μghA20-pro-2-PDox.

FIG. 9. Effect of different single doses of hRS7-pro-2-PDox on growth ofhuman gastric carcinoma xenografts.

FIG. 10. Effect of different single doses of hRS7-pro-2-PDox on survivalof mice bearing human gastric carcinoma xenografts.

FIG. 11. ADCC of various hRS7-ADCs vs. hRS7 IgG.

FIG. 12A. Structures of CL2-SN-38 and CL2A-SN-38.

FIG. 12B. Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs. CL2Alinkers versus hA20 ADC and saline control, using COLO 205 colonicadenocarcinoma. Animals were treated twice weekly for 4 weeks asindicated by the arrows. COLO 205 mice (N=6) were treated with 0.4 mg/kgADC and tumors measured twice a week.

FIG. 12C. Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs. CL2Alinkers versus hA20 ADC and saline control, using Capan-1 pancreaticadenocarcinoma. Animals were treated twice weekly for 4 weeks asindicated by the arrows. Capan-1 mice (N=10) were treated with 0.2 mg/kgADC and tumors measured weekly.

FIG. 13A. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Mice bearing Calu-3tumors (N=5-7) were injected with hRS7-CL2-SN-38 every 4 days for atotal of 4 injections (q4d×4).

FIG. 13B. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). COLO 205 tumor-bearingmice (N=5) were injected 8 times (q4d×8) with the ADC or every 2 daysfor a total of 5 injections (q2d×5) with the MTD of irinotecan.

FIG. 13C. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Capan-1 (N=10) weretreated twice weekly for 4 weeks with the agents indicated.

FIG. 13D. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). BxPC-3 tumor-bearingmice (N=10) were treated twice weekly for 4 weeks with the agentsindicated.

FIG. 13E. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). In addition to ADCgiven twice weekly for 4 week, SK-MES-1 tumor-bearing (N=8) micereceived the MTD of CPT-11 (q2d×5).

FIG. 14A. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of AST.

FIG. 14B. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of ALT.

FIG. 14C. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in neutrophil counts in Cynomolgusmonkeys.

FIG. 14D. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in platelet counts in Cynomolgusmonkeys.

FIG. 15. In vitro efficacy of anti-Trop-2-paclitaxel ADC againstMDA-MB-468 human breast adenocarcinoma.

FIG. 16. In vitro efficacy of anti-Trop-2-paclitaxel ADC against BxPC-3human pancreatic adenocarcinoma.

FIG. 17A. Comparison of in vitro efficacy of anti-Trop-2 ADCs(hRS7-SN-38 versus MAB650-SN-38) in Capan-1 human pancreaticadenocarcinoma.

FIG. 17B. Comparison of in vitro efficacy of anti-Trop-2 ADCs(hRS7-SN-38 versus MAB650-SN-38) in BxPC-3 human pancreaticadenocarcinoma.

FIG. 17C. Comparison of in vitro efficacy of anti-Trop-2 ADCs(hRS7-SN-38 versus MAB650-SN-38) in NCI-N87 human gastricadenocarcinoma.

FIG. 18A. Comparison of cytotoxicity of naked or SN-38 conjugated hRS7vs. 162-46.2 antibodies in BxPC-3 human pancreatic adenocarcinoma.

FIG. 18B. Comparison of cytotoxicity of naked or SN-38 conjugated hRS7vs. 162-46.2 antibodies in MDA-MB-468 human breast adenocarcinoma.

FIG. 19. IMMU-132 phase I/II data for best response by RECIST criteria.

FIG. 20. IMMU-132 phase I/II data for time to progression and bestresponse (RECIST).

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Antibody fragments may also include singledomain antibodies and IgG4 half-molecules, as discussed below.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody. The term“antibody fragment” also includes isolated fragments consisting of thevariable regions of antibodies, such as the “Fv” fragments consisting ofthe variable regions of the heavy and light chains and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (“scFv proteins”).

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples section of which are incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptoticagents, anti-angiogenic agents, boron compounds, photoactive agents ordyes and radioisotopes. Therapeutic agents of use are described in moredetail below.

An immunoconjugate is an antibody, antibody fragment or fusion proteinconjugated to at least one therapeutic and/or diagnostic agent.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. Multispecific, multivalentantibodies are constructs that have more than one binding site, and thebinding sites are of different specificity.

A bispecific antibody is an antibody that can bind simultaneously to twodifferent targets. Bispecific antibodies (bsAb) and bispecific antibodyfragments (bsFab) may have at least one arm that specifically binds to,for example, a tumor-associated antigen and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering.

Anti-Trop-2 Antibodies

The subject ADCs may include an antibody or fragment thereof that bindsto Trop-2. In a specific preferred embodiment, the anti-Trop-2 antibodymay be a humanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785,incorporated herein by reference in its entirety), comprising the lightchain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful forimmunotoxin production. (Id.) It is well documented that internalizationof immunotoxin conjugates is a requirement for anti-tumor activity.(Pastan et al., Cell 47:641, 1986) Internalization of drugimmunoconjugates has been described as a major factor in anti-tumorefficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1189, 1988) Thus,the RS7 antibody exhibits several important properties for therapeuticapplications.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject ADCs. While humanized or human antibodies arepreferred for reduced immunogenicity, in alternative embodiments achimeric antibody may be of use. As discussed below, methods of antibodyhumanization are well known in the art and may be utilized to convert anavailable murine or chimeric antibody into a humanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. U.S. Pat. No. 8,715,662discloses anti-Trop-2 antibodies produced by hybridomas deposited at theAID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD08021. U.S. Patent Application Publ. No. 20120237518 disclosesanti-Trop-2 antibodies 77220, KM4097 and KM4590. U.S. Pat. No. 8,309,094(Wyeth) discloses antibodies A1 and A3, identified by sequence listing.The Examples section of each patent or patent application cited above inthis paragraph is incorporated herein by reference. Non-patentpublication Lipinski et al. (1981, Proc Natl. Acad Sci USA, 78:5147-50)disclosed anti-Trop-2 antibodies 162-25.3 and 162-46.2.

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. The sequence of the human Trop-2protein was also known in the art (see, e.g., GenBank Accession No.CAA54801.1). Methods for producing humanized, human or chimericantibodies were also known. The person of ordinary skill, reading theinstant disclosure in light of general knowledge in the art, would havebeen able to make and use the genus of anti-Trop-2 antibodies in thesubject ADCs.

Use of anti-Trop-2 antibodies has been disclosed for immunotherapeuticsother than ADCs. The murine IgG2a antibody edrecolomab (PANOREX®) hasbeen used for treatment of colorectal cancer, although the murineantibody is not well suited for human clinical use (Baeuerle & Gires,2007, Br. J Cancer 96:417-423). Low-dose subcutaneous administration ofecrecolomab was reported to induce humoral immune responses against thevaccine antigen (Baeuerle & Gires, 2007). Adecatumumab (MT201), a fullyhuman anti-Trop-2 antibody, has been used in metastatic breast cancerand early-stage prostate cancer and is reported to act through ADCC andCDC activity (Baeuerle & Gires, 2007). MT110, a single-chainanti-Trop-2/anti-CD3 bispecific antibody construct has reported efficacyagainst ovarian cancer (Baeuerle & Gires, 2007). Catumaxomab, a hybridmouse/rat antibody with binding affinity for Trop-2, CD3 and Fcreceptor, was reported to be active against ovarian cancer (Baeuerle &Gires, 2007). Proxinium, an immunotoxin comprising anti-Trop-2single-chain antibody fused to Pseudomonas exotoxin, has been tested inhead-and-neck and bladder cancer (Baeuerle & Gires, 2007). None of thesestudies contained any disclosure of the use of anti-Trop-2 antibody-drugconjugates.

Anti-CEA Antibodies

Certain embodiments may concern use of conjugated antibodies againstCEACAM5 or CEACAM6. CEA (CEACAM5) is an oncofetal antigen commonlyexpressed in a number of epithelial cancers, most commonly those arisingin the colon but also in the breast, lung, pancreas, thyroid (medullarytype) and ovary (Goldenberg et al., J. Natl. Cancer Inst. 57: 11-22,1976; Shively, et al., Crit. Rev. Oncol. Hematol. 2:355-399, 1985). Thehuman CEA gene family is composed of 7 known genes belonging to theCEACAM subgroup. These subgroup members are mainly associated with thecell membrane and show a complex expression pattern in normal andcancerous tissues. The CEACAM5 gene, also known as CD66e, codes for theCEA protein (Beauchemin et al., Exp Cell Res 252:243, 1999). CEACAM5 wasfirst described in 1965 as a gastrointestinal oncofetal antigen (Gold etal., J Exp Med 122:467-481, 1965), but is now known to be overexpressedin a majority of carcinomas, including those of the gastrointestinaltract, the respiratory and genitourinary systems, and breast cancer(Goldenberg et al., J Natl Cancer Inst. 57:11-22, 1976; Shively andBeatty, Crit Rev Oncol Hematol 2:355-99, 1985).

CEACAM6 (also called CD66c or NCA-90) is a non-specific cross-reactingglycoprotein antigen that shares some, but not all, antigenicdeterminants with CEACAM5 (Kuroki et al., Biochem Biophys Res Comm182:501-06, 1992). CEACAM6 is expressed on granulocytes and epitheliafrom various organs, and has a broader expression zone in proliferatingcells of hyperplastic colonic polyps and adenomas, compared with normalmucosa, as well as by many human cancers (Scholzel et al., Am J Pathol157:1051-52, 2000; Kuroki et al., Anticancer Res 19:5599-5606, 1999).Relatively high serum levels of CEACAM6 are found in patients with lung,pancreatic, breast, colorectal, and hepatocellular carcinomas. Theamount of CEACAM6 does not correlate with the amount of CEACAM5expressed (Kuroki et al., Anticancer Res 19:5599-5606, 1999).

Expression of CEACAM6 in colorectal cancer correlates inversely withcellular differentiation (Ilantzis et al., Neoplasia 4:151-63, 2002) andis an independent prognostic factor associated with a higher risk ofrelapse (Jantscheff et al., J Clin Oncol 21:3638-46, 2003). Both CEACAM5and CEACAM6 have a role in cell adhesion, invasion and metastasis.CEACAM5 has been shown to be involved in both homophilic (CEA to CEA)and heterophilic (CEA binding to non-CEA molecules) interactions(Bechimol et al., Cell 57:327-34, 1989; Oikawa et al., Biochem BiophysRes Comm 164:39-45, 1989), suggesting to some that it is anintercellular adhesion molecule involved in cancer invasion andmetastasis (Thomas et al., Cancer Lett 92:59-66, 1995). These reactionswere completely inhibited by the Fab′ fragment of an anti-CEACAM5antibody (Oikawa et al., Biochem Biophys Res Comm 164:39-45, 1989).CEACAM6 also exhibits homotypic binding with other members of the CEAfamily and heterotypic interactions with integrin receptors (Stannersand Fuks, In: Cell Adhesion and Communication by the CEA Family,(Stanners ed.) Vol. 5, pp. 57-72, Harwood Academic Publ., Amsterdam,1998). Antibodies that target the N-domain of CEACAM6 interfere withcell-cell interactions (Yamanka et al. Biochem Biophys Res Comm219:842-47, 1996). Many breast, pancreatic, colonic and non-small-celllung cancer (NSCLC) cell lines express CEACAM6 and anti-CEACAM6 antibodyinhibits in vitro migration, invasion, and adhesion of antigen-positivecells (Blumenthal et al, Cancer Res 65:8809-17, 2005).

Anti-CEA antibodies are classified into different categories, dependingon their cross-reactivity with antigens other than CEA. Anti-CEAantibody classification was described by Primus and Goldenberg, U.S.Pat. No. 4,818,709 (incorporated herein by reference from Col. 3, line 5through Col. 26, line 49). The classification of anti-CEA antibodies isdetermined by their binding to CEA, meconium antigen (MA) andnonspecific crossreacting antigen (NCA). Class I anti-CEA antibodiesbind to all three antigens. Class II antibodies bind to MA and CEA, butnot to NCA. Class III antibodies bind only to CEA (U.S. Pat. No.4,818,709). Examples of each class of anti-CEA antibody are known, suchas MN-3, MN-15 and NP-1 (Class I); MN-2, NP-2 and NP-3 (Class II); andMN-14 and NP-4 (Class III) (U.S. Pat. No. 4,818,709; Blumenthal et al.BMC Cancer 7:2 (2007)).

The epitopic binding sites of various anti-CEA antibodies have also beenidentified. The MN-15 antibody binds to the A1B1 domain of CEA, the MN-3antibody binds to the N-terminal domain of CEA and the MN-14 antibodybinds to the A3B3 (CD66e) domain of CEA (Blumenthal et al. BMC Cancer7:2 (2007)). There is no direct correlation between epitopic bindingsite and class of anti-CEA antibody. For example, MN-3 and MN-15 areboth Class I anti-CEA antibodies, reactive with NCA, MA and CEA, butbind respectively to the N-terminal and A1B1 domains of CEA. Primus andGoldenberg (U.S. Pat. No. 4,818,709) reported a complicated pattern ofcross-blocking activity between the different anti-CEA antibodies, withNP-1 (Class I) and NP-2 (Class II) cross-blocking binding to CEA of eachother, but neither blocking binding of NP-3 (Class II). However, bydefinition Class I anti-CEA antibodies bind to both CEACAM5 and CEACAM6,while Class III anti-CEA antibodies bind only to CEACAM5.

Anti-CEA antibodies have been suggested for therapeutic treatment of avariety of cancers. For example, medullary thyroid cancer (MTC) confinedto the thyroid gland is generally treated by total thyroidectomy andcentral lymph node dissection. However, disease recurs in approximately50% of these patients. In addition, the prognosis of patients withunresectable disease or distant metastases is poor, less than 30%survive 10 years (Rossi et al., Amer. J. Surgery, 139:554 (1980); Samaanet al., J. Clin. Endocrinol. Metab., 67:801 (1988); Schroder et al.,Cancer, 61:806 (1988)). These patients are left with few therapeuticchoices (Principles and Practice of Oncology, DeVita, Hellman andRosenberg (eds.), New York: JB Lippincott Co., pp. 1333-1435 (1989);Cancer et al., Current Problems Surgery, 22: 1 (1985)). The Class IIIanti-CEA antibody MN-14 has been reported to be effective for therapy ofhuman medullary thyroid carcinoma in an animal xenograft model system,when used in conjunction with pro-apoptotic agents such as DTIC, CPT-11and 5-fluorouracil (U.S. patent application Ser. No. 10/680,734, theExamples section of which is incorporated herein by reference). TheClass III anti-CEA antibody reportedly sensitized cancer cells totherapy with chemotherapeutic agents and the combination of antibody andchemotherapeutic agent was reported to have synergistic effects ontumors compared with either antibody or chemotherapeutic agent alone(U.S. Ser. No. 10/680,734). Anti-CEA antibodies of different classes(such as MN-3, MN-14 and MN-15) have been proposed for use in treating avariety of tumors.

In a preferred embodiment, therapeutic conjugates comprising ananti-CEACAM5 antibody (e.g., hMN-14, labretuzumab) and/or ananti-CEACAM6 antibody (e.g., hMN-3 or hMN-15) may be used to treat anyof a variety of cancers that express CEACAM5 and/or CEACAM6, asdisclosed in U.S. Pat. Nos. 7,541,440; 7,951,369; 5,874,540; 6,676,924and 8,267,865, the Examples section of each incorporated herein byreference. Solid tumors that may be treated using anti-CEACAM5,anti-CEACAM6, or a combination of the two include but are not limited tobreast, lung, pancreatic, esophageal, medullary thyroid, ovarian, colon,rectum, urinary bladder, mouth and stomach cancers. A majority ofcarcinomas, including gastrointestinal, respiratory, genitourinary andbreast cancers express CEACAM5 and may be treated with the subjectimmunoconjugates. An hMN-14 antibody is a humanized antibody thatcomprises light chain variable region CDR sequences CDR1 (KASQDVGTSVA;SEQ ID NO:114), CDR2 (WTSTRHT; SEQ ID NO:97), and CDR3 (QQYSLYRS; SEQ IDNO:98), and the heavy chain variable region CDR sequences CDR1 (TYWMS;SEQ ID NO:99), CDR2 (EIHPDSSTINYAPSLKD; SEQ ID NO:100) and CDR3(LYFGFPWFAY; SEQ ID NO:101). An hMN-3 antibody is a humanized antibodythat comprises light chain variable region CDR sequences CDR1(RSSQSIVHSNGNTYLE, SEQ ID NO:102), CDR2 (KVSNRFS, SEQ ID NO:103) andCDR3 (FQGSHVPPT, SEQ ID NO: 104) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:105), CDR2 (WINTYTGEPTYADDFKG, SEQ ID NO:106) and CDR3(KGWMDFNSSLDY, SEQ ID NO:107). An hMN-15 antibody is a humanizedantibody that comprises light chain variable region CDR sequencesSASSRVSYIH (SEQ ID NO:108); GTSTLAS (SEQ ID NO:109); and QQWSYNPPT (SEQID NO:110); and heavy chain variable region CDR sequences DYYMS (SEQ IDNO:111); FIANKANGHTTDYSPSVKG (SEQ ID NO:112); and DMGIRWNFDV (SEQ IDNO:113).

Although use of MN-14, MN-15 or MN-3 is preferred, other antibodiesagainst CEACAM5 or CEACAM6 are known in the art and may be utilized asimmunoconjugates, such as SN-38 conjugates. Another exemplary antibodyagainst CEACAM5 is the anti-CEACAM5 CC4 antibody (e.g., Zheng et al.,2011, PLoS One 6:e21146). Antibodies against CEACAM5 or CEACAM6 areavailable from numerous commercial sources, including LS-C6031,LS-B7292, LS-C338757 (LSBio, Seattle, Wash.); SAB1307198, GW22478,HPA019758 (Sigma-Aldrich, St. Louis, Mo.); sc-23928, sc-59872, sc-52390(Santa Cruz Biotechnology, Santa Cruz, Calif.); and ab78029 (ABCAM®,Cambridge, Mass.). Any such known anti-CEACAM5 or anti-CEACAM6 antibodymay be used in the immunoconjugates disclosed herein.

Antibody Preparation

Techniques for preparing monoclonal antibodies against virtually anytarget antigen, such as Trop-2 or CEACAM5, are well known in the art.See, for example, Köhler and Milstein, Nature 256: 495 (1975), andColigan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,removing the spleen to obtain B-lymphocytes, fusing the B-lymphocyteswith myeloma cells to produce hybridomas, cloning the hybridomas,selecting positive clones which produce antibodies to the antigen,culturing the clones that produce antibodies to the antigen, andisolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies and Target Antigens

As discussed above, in preferred embodiments the ADCs are of use fortreatment of cancer. In certain embodiments, the target cancer mayexpress one or more target tumor-associated antigens (TAAs). Particularantibodies that may be of use for therapy of cancer include, but are notlimited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20,anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20),lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor),ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1,also known as Trop-2)), PAM4 or KC4 (both anti-mucin), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5),MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu31 (an anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19),TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specificmembrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA),G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-mucin) andtrastuzumab (anti-ErbB2). Such antibodies are known in the art (e.g.,U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393;6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403;7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655;7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. Patent ApplicationPubl. No. 20050271671; 20060193865; 20060210475; 20070087001; theExamples section of each incorporated herein by reference.) Specificknown antibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20(U.S. Pat. No. 7,151,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU-31(U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318), hLL2 (U.S.Pat. No. 5,789,554), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat.No. 7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.7,541,440), hR1 (U.S. patent application Ser. No. 12/772,645), hRS7(U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440),AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372, depositedas ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575) the text ofeach recited patent or application is incorporated herein by referencewith respect to the Figures and Examples sections.

Other useful tumor-associated antigens that may be targeted includecarbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1,CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20(e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55,CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133,CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein (AFP),VEGF (e.g., AVASTIN®, fibronectin splice variant), ED-B fibronectin(e.g., L19), EGP-1 (Trop-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1)(e.g., ERBITUX®), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor,Ga 733, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24,HER-2/neu, histone H2B, histone H3, histone H4, insulin-like growthfactor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigento which L243 binds, CD66 antigens, i.e., CD66a-d or a combinationthereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophagemigration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac,placental growth factor (P1GF), PSA (prostate-specific antigen), PSMA,PAM4 antigen, PD-1 receptor, PD-L, NCA-95, NCA-90, A3, A33, Ep-CAM,KS-1, Le(y), mesothelin, S100, tenascin, TAC, Tn antigen,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), Trop-2, VEGFR,RANTES, T101, as well as cancer stem cell antigens, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

Cancer stem cells, which are ascribed to be more therapy-resistantprecursor malignant cell populations (Hill and Perris, J. Natl. CancerInst. 2007; 99:1435-40), have antigens that can be targeted in certaincancer types, such as CD133 in prostate cancer (Maitland et al., ErnstSchering Found. Sympos. Proc. 2006; 5:155-79), non-small-cell lungcancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91), andglioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5), and CD44 incolorectal cancer (Dalerba er al., Proc. Natl. Acad. Sci. USA 2007;104(24)10158-63), pancreatic cancer (Li et al., Cancer Res. 2007;67(3):1030-7), and in head and neck squamous cell carcinoma (Prince etal., Proc. Natl. Acad. Sci. USA 2007; 104(3)973-8). Another usefultarget for breast cancer therapy is the LIV-1 antigen described byTaylor et al. (Biochem. J. 2003; 375:51-9).

Checkpoint inhibitor antibodies have been used in cancer therapy. Immunecheckpoints refer to inhibitory pathways in the immune system that areresponsible for maintaining self-tolerance and modulating the degree ofimmune system response to minimize peripheral tissue damage. However,tumor cells can also activate immune system checkpoints to decrease theeffectiveness of immune response against tumor tissues. Exemplarycheckpoint inhibitor antibodies against cytotoxic T-lymphocyte antigen 4(CTLA4, also known as CD152), programmed cell death protein 1 (PD1, alsoknown as CD279) and programmed cell death 1 ligand 1 (PD-L1, also knownas CD274), may be used in combination with one or more other agents toenhance the effectiveness of immune response against disease cells,tissues or pathogens. Exemplary anti-PD 1 antibodies includelambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERSSQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011, CURETECH LTD.).Anti-PD1 antibodies are commercially available, for example from ABCAM®(AB137132), BIOLEGEND® (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE(J105, J116, MIH4). Exemplary anti-PD-L1 antibodies include MDX-1105(MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559(BRISTOL-MYERS SQUIBB). Anti-PD-L1 antibodies are also commerciallyavailable, for example from AFFYMETRIX EBIOSCIENCE (MIH1). Exemplaryanti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) andtremelimumab (PFIZER). Anti-PD1 antibodies are commercially available,for example from ABCAM® (AB134090), SINO BIOLOGICAL INC. (11159-H03H,11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967,PA5-26465, MA1-12205, MA1-35914). Ipilimumab has recently received FDAapproval for treatment of metastatic melanoma (Wada et al., 2013, JTransl Med 11:89).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, and colon (e.g., Meyer-Siegler et al.,2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma52:1446-54). Milatuzumab (hLL1) is an exemplary anti-CD74 antibody oftherapeutic use for treatment of MIF-mediated diseases.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.)

Antibodies of use may be commercially obtained from a wide variety ofknown sources.

For example, a variety of antibody secreting hybridoma lines areavailable from the American Type Culture Collection (ATCC, Manassas,Va.). A large number of antibodies against various disease targets,including tumor-associated antigens, have been deposited at the ATCCand/or have published variable region sequences and are available foruse in the claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206′ 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338. These are exemplary only anda wide variety of other antibodies and their hybridomas are known in theart. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown belowfor the exemplary antibodies rituximab (SEQ ID NO:7) and veltuzumab (SEQID NO:8).

Rituximab heavy chain variable region sequence (SEQ ID NO: 7)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVeltuzumab heavy chain variable region (SEQ ID NO: 8)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Nanobodies

Nanobodies are single-domain antibodies of about 12-15 kDa in size(about 110 amino acids in length). Nanobodies can selectively bind totarget antigens, like full-size antibodies, and have similar affinitiesfor antigens. However, because of their much smaller size, they may becapable of better penetration into solid tumors. The smaller size alsocontributes to the stability of the nanobody, which is more resistant topH and temperature extremes than full size antibodies (Van Der Linden etal., 1999, Biochim Biophys Act 1431:37-46). Single-domain antibodieswere originally developed following the discovery that camelids (camels,alpacas, llamas) possess fully functional antibodies without lightchains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77:13-22).The heavy-chain antibodies consist of a single variable domain (V_(HH))and two constant domains (C_(H)2 and C_(H)3). Like antibodies,nanobodies may be developed and used as multivalent and/or bispecificconstructs. Humanized forms of nanobodies are in commercial developmentthat are targeted to a variety of target antigens, such as IL-6R, vWF,TNF, RSV, RANKL, IL-17A & F and IgE (e.g., ABLYNX®, Ghent, Belgium),with potential clinical use in cancer and other disorders (e.g., Saerenset al., 2008, Curr Opin Pharmacol 8:600-8; Muyldermans, 2013, Ann RevBiochem 82:775-97; Ibanez et al., 2011, J Infect Dis 203:1063-72).

The plasma half-life of nanobodies is shorter than that of full-sizeantibodies, with elimination primarily by the renal route. Because theylack an Fc region, they do not exhibit complement dependentcytotoxicity.

Nanobodies may be produced by immunization of camels, llamas, alpacas orsharks with target antigen, following by isolation of mRNA, cloning intolibraries and screening for antigen binding. Nanobody sequences may behumanized by standard techniques (e.g., Jones et al., 1986, Nature 321:522, Riechmann et al., 1988, Nature 332: 323, Verhoeyen et al., 1988,Science 239: 1534, Carter et al., 1992, Proc. Nat'l Acad. Sci. USA 89:4285, Sandhu, 1992, Crit. Rev. Biotech. 12: 437, Singer et al., 1993, J.Immun. 150: 2844). Humanization is relatively straight-forward becauseof the high homology between camelid and human FR sequences.

In various embodiments, the subject ADCs may comprise nanobodies fortargeted delivery of conjugated drug to targeted cancer cells.Nanobodies of use are disclosed, for example, in U.S. Pat. Nos.7,807,162; 7,939,277; 8,188,223; 8,217,140; 8,372,398; 8,557,965;8,623,361 and 8,629,244, the Examples section of each incorporatedherein by reference.)

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies. (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 kDfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

General Techniques for Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding V_(κ) (variable light chain) and V_(H) (variableheavy chain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The V_(κ) sequence for the MAb may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for V_(κ) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the V_(κ) andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the V_(κ) andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Bispecific and Multispecific Antibodies

In certain embodiments the ADC and one or more other therapeuticantibodies may be administered as separate antibodies, eithersequentially or concurrently. In alternative embodiments, antibodies orantibody fragments may be administered as a single bispecific ormultispecific antibody. Numerous methods to produce bispecific ormultispecific antibodies are known, as disclosed, for example, in U.S.Pat. No. 7,405,320, the Examples section of which is incorporated hereinby reference. Bispecific antibodies can be produced by the quadromamethod, which involves the fusion of two different hybridomas, eachproducing a monoclonal antibody recognizing a different antigenic site(Milstein and Cuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Other methods include improving theefficiency of generating hybrid hybridomas by gene transfer of distinctselectable markers via retrovirus-derived shuttle vectors intorespective parental hybridomas, which are fused subsequently (DeMonte,et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of ahybridoma cell line with expression plasmids containing the heavy andlight chain genes of a different antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv), as discussedabove. Reduction of the peptide linker length to less than 12 amino acidresidues prevents pairing of V_(H) and V_(L) domains on the same chainand forces pairing of V_(H) and V_(L) domains with complementary domainson other chains, resulting in the formation of functional multimers.Polypeptide chains of V_(H) and V_(L) domains that are joined withlinkers between 3 and 12 amino acid residues form predominantly dimers(termed diabodies). With linkers between 0 and 2 amino acid residues,trimers (termed triabody) and tetramers (termed tetrabody) are favored,but the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of V-domains (V_(H)-linker-V_(L)or V_(L)-linker-V_(H)), in addition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “DOCK-AND-LOCK™” (DNL™), discussedin more detail below, has been utilized to produce combinations ofvirtually any desired antibodies, antibody fragments and other effectormolecules. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

Dock-and-Lock™ (DNL™)

Bispecific or multispecific antibodies or other constructs may beproduced using the DOCK-AND-LOCK™ technology (see, e.g., U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, the Examplessection of each incorporated herein by reference). Generally, thetechnique takes advantage of the specific and high-affinity bindinginteractions that occur between a dimerization and docking domain (DDD)sequence of the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and an anchor domain (AD) sequence derived from any of a varietyof AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wongand Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and ADpeptides may be attached to any protein, peptide or other molecule.Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the technique allows the formation of complexes between anyselected molecules that may be attached to DDD or AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R . . . subunits (RI and RII), andeach type has a and P3 isoforms (Scott, Pharmacol. Ther. 1991; 50:123).Thus, the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIαand RIIβ. The R subunits have been isolated only as stable dimers andthe dimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described below, virtually any proteinor peptide may be incorporated into a DNL™ construct. However, thetechnique is not limiting and other methods of conjugation may beutilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 9) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 10) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 11) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 12)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 13) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERL EKEEAKDDD3C (SEQ ID NO: 14) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 15) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 16) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 17)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKL EKEENRQILA PKA RIIα(SEQ ID NO: 18) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 19) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:9 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 9) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:9are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:20 to SEQ ID NO:39 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2 Conservative Amino Acid Substitutionsin DDD1 (SEQ ID NO: 9). Consensus sequence disclosed as SEQ ID NO: 94. SH I Q I P P G L T E L L Q G Y T V E V L R T K N A S D N A S D K R Q Q PP D L V E F A V E Y F T R L R E A R A N N E D L D S K K D L K L I I I VV V (SEQ ID NO: 20) THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 21) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 22) SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 23) SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 24) SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 25) SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 26) SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 27) SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 28) SHIQ1PPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 29) SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 30) SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 31) SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 32) SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ ID NO: 33) SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ ID NO: 34) SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ ID NO: 35) SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ ID NO: 36) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ ID NO: 37) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ ID NO: 38) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ ID NO: 39) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO: 11), witha binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designedas a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO: 11 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:19), similar tothat shown for DDD1 (SEQ ID NO:16) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:40 to SEQ ID NO:57 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 11) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutionsin AD1 (SEQ ID NO: 11). Consensus sequence disclosed as SEQ ID NO: 95. QI E Y L A K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S V(SEQ ID NO: 40) NIEYLAKQIVDNAIQQA (SEQ ID NO: 41) QLEYLAKQIVDNAIQQA(SEQ ID NO: 42) QVEYLAKQIVDNAIQQA (SEQ ID NO: 43) QIDYLAKQIVDNAIQQA(SEQ ID NO: 44) QIEFLAKQIVDNAIQQA (SEQ ID NO: 45) QIETLAKQIVDNAIQQA(SEQ ID NO: 46) QIESLAKQIVDNAIQQA (SEQ ID NO: 47) QIEYIAKQIVDNAIQQA(SEQ ID NO: 48) QIEYVAKQIVDNAIQQA (SEQ ID NO: 49) QIEYLARQIVDNAIQQA(SEQ ID NO: 50) QIEYLAKNIVDNAIQQA (SEQ ID NO: 51) QIEYLAKQIVENAIQQA(SEQ ID NO: 52) QIEYLAKQIVDQAIQQA (SEQ ID NO: 53) QIEYLAKQIVDNAINQA(SEQ ID NO: 54) QIEYLAKQIVDNAIQNA (SEQ ID NO: 55) QIEYLAKQIVDNAIQQL(SEQ ID NO: 56) QIEYLAKQIVDNAIQQI (SEQ ID NO: 57) QIEYLAKQIVDNAIQQV

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:58),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:59-61.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:12, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 58) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 59) QIEYKAKQIVDHAIHQA(SEQ ID NO: 60) QIEYHAKQIVDHAIHQA (SEQ ID NO: 61) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 62) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 63) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 64)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 65)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 66) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 67) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 68) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 69)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 70) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 71) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:72-74. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:72), RIAD (SEQ IDNO:73) and PV-38 (SEQ ID NO:74). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 72) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 73)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 74) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 11) AKAPIS-P QIEYLAKQLPDNAIQQA (SEQ ID NO: 75) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 76) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 77) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 78) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 79) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 80) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 81) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 82) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 83) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 84) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 85) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 86) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 87) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 88) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 89) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 90) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 91) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 92)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO: 11). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 11) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:9. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 9) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:9) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5 Conservative Amino Acid Substitutionsin DDD1 (SEQ ID NO: 9). Consensus sequence disclosed as SEQ ID NO: 96. SH I Q I P P G L T E L L Q G Y T V E V L R T N S I L A Q Q P P D L V E FA V E Y F T R L R E A R A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Alternative DNL™ Structures

In certain alternative embodiments, DNL™ constructs may be formed usingalternatively constructed antibodies or antibody fragments, in which anAD moiety may be attached at the C-terminal end of the kappa light chain(C_(k)), instead of the C-terminal end of the Fc on the heavy chain. Thealternatively formed DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. No. 61/654,310, filed Jun. 1,2012, 61/662,086, filed Jun. 20, 2012, 61/673,553, filed Jul. 19, 2012,and 61/682,531, filed Aug. 13, 2012, the entire text of eachincorporated herein by reference. The light chain conjugated DNL™constructs exhibit enhanced Fc-effector function activity in vitro andimproved pharmacokinetics, stability and anti-lymphoma activity in vivo(Rossi et al., 2013, Bioconjug Chem 24:63-71).

C_(k)-conjugated DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. No. 61/654,310, 61/662,086,61/673,553, and 61/682,531. Briefly, C_(k)-AD2-IgG, was generated byrecombinant engineering, whereby the AD2 peptide was fused to theC-terminal end of the kappa light chain. Because the natural C-terminusof C_(K) is a cysteine residue, which forms a disulfide bridge to CH1, a16-amino acid residue “hinge” linker was used to space the AD2 from theC_(K)-V_(H)1 disulfide bridge. The mammalian expression vectors forC_(k)-AD2-IgG-veltuzumab and C_(k)-AD2-IgG-epratuzumab were constructedusing the pdHL2 vector, which was used previously for expression of thehomologous C_(H)3-AD2-IgG modules. A 2208-bp nucleotide sequence wassynthesized comprising the pdHL2 vector sequence ranging from the Bam HIrestriction site within the V_(K)/C_(K) intron to the XhoI restrictionsite 3′ of the C_(k) intron, with the insertion of the coding sequencefor the hinge linker (EFPKPSTPPGSSGGAP, SEQ ID NO:93) and AD2, in frameat the 3′end of the coding sequence for C_(K). This synthetic sequencewas inserted into the IgG-pdHL2 expression vectors for veltuzumab andepratuzumab via Bam HI and Xho I restriction sites. Generation ofproduction clones with SpESFX-10 were performed as described for theC_(H)3-AD2-IgG modules. C_(k)-AD2-IgG-veltuzumab andC_(k)-AD2-IgG-epratuzumab were produced by stably-transfected productionclones in batch roller bottle culture, and purified from the supernatantfluid in a single step using MabSelect (GE Healthcare) Protein Aaffinity chromatography.

Following the same DNL™ process described previously for 22-(20)-(20)(Rossi et al., 2009, Blood 113:6161-71), C_(k)-AD2-IgG-epratuzumab wasconjugated with C_(H)1-DDD2-Fab-veltuzumab, a Fab-based module derivedfrom veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22*indicates the C_(k)-AD2 module of epratuzumab and each (20) symbolizes astabilized dimer of veltuzumab Fab. The properties of 22*-(20)-(20) werecompared with those of 22-(20)-(20), the homologous Fc-bsHexAbcomprising C_(H)3-AD2-IgG-epratuzumab, which has similar composition andmolecular size, but a different architecture.

Following the same DNL™ process described previously for 20-2b (Rossi etal., 2009, Blood 114:3864-71), C_(k)-AD2-IgG-veltuzumab, was conjugatedwith IFNα2b-DDD2, a module of IFNα2b with a DDD2 peptide fused at itsC-terminal end, to generate 20*-2b, which comprises veltuzumab with adimeric IFNα2b fused to each light chain. The properties of 20*-2b werecompared with those of 20-2b, which is the homologous Fc-IgG-IFNα.

Each of the bsHexAbs and IgG-IFNα were isolated from the DNL™ reactionmixture by MabSelect affinity chromatography. The two C_(k)-derivedprototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprisingepratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and aCD20-targeting immunocytokine, comprising veltuzumab and four moleculesof interferon-α2b, displayed enhanced Fc-effector functions in vitro, aswell as improved pharmacokinetics, stability and anti-lymphoma activityin vivo, compared to their Fc-derived counterparts.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Pre-Targeting

Bispecific or multispecific antibodies may be of use in pretargetingtechniques. In this case, one or more therapeutic agent may beconjugated to a targetable construct that comprises one or more haptens.The hapten is recognized by at least one arm of a bispecific ormultispecific antibody that also binds to a tumor-associated antigen orother disease-associated antigen. In this case, the therapeutic agentbinds indirectly to the antibodies, via the binding of the targetableconstruct. This process is referred to as pretargeting.

Pre-targeting is a multistep process originally developed to resolve theslow blood clearance of directly targeting antibodies, which contributesto undesirable toxicity to normal tissues such as bone marrow. Withpre-targeting, a therapeutic agent is attached to a small deliverymolecule (targetable construct) that is cleared within minutes from theblood. A pre-targeting bispecific or multispecific antibody, which hasbinding sites for the targetable construct as well as a target antigen,is administered first, free antibody is allowed to clear fromcirculation and then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Targetable Constructs

In certain embodiments, targetable construct peptides labeled with oneor more therapeutic or diagnostic agents for use in pre-targeting may beselected to bind to a bispecific antibody with one or more binding sitesfor a targetable construct peptide and one or more binding sites for atarget antigen associated with a disease or condition. Bispecificantibodies may be used in a pretargeting technique wherein the antibodymay be administered first to a subject. Sufficient time may be allowedfor the bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues and one or more haptens for binding, e.g., to abispecific antibody. Exemplary haptens may include In-DTPA(indium-diethylene triamine pentaacetic acid) or HSG (histamine succinylglycine). The targetable construct may also comprise one or morechelating moieties, such as DOTA(1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid), NOTA(1,4,7-triaza-cyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties. Chelating moieties may be used,for example, to bind to a therapeutic and or diagnostic radionuclide,paramagnetic ion or contrast agent.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

Immunoconjugates

In certain embodiments, a cytotoxic drug or other therapeutic ordiagnostic agent may be covalently attached to an antibody or antibodyfragment to form an immunoconjugate. In some embodiments, a drug orother agent may be attached to an antibody or fragment thereof via acarrier moiety. Carrier moieties may be attached, for example to reducedSH groups and/or to carbohydrate side chains. A carrier moiety can beattached at the hinge region of a reduced antibody component viadisulfide bond formation. Alternatively, such agents can be attachedusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et. al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of the ADC is anantibody fragment. However, it is possible to introduce a carbohydratemoiety into the light chain variable region of a full length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples section ofwhich is incorporated herein by reference. The engineered carbohydratemoiety is used to attach the therapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule.

In the presence of a second molecule comprising an azide moiety, theazide reacts with the activated alkyne to form a 1,4-disubstituted1,2,3-triazole. The copper catalyzed reaction occurs at room temperatureand is sufficiently specific that purification of the reaction productis often not required. (Rostovstev et al., 2002, Angew Chem Int Ed41:2596; Tornoe et al., 2002, J Org Chem 67:3057.) The azide and alkynefunctional groups are largely inert towards biomolecules in aqueousmedium, allowing the reaction to occur in complex solutions. Thetriazole formed is chemically stable and is not subject to enzymaticcleavage, making the click chemistry product highly stable in biologicalsystems. Although the copper catalyst is toxic to living cells, thecopper-based click chemistry reaction may be used in vitro forimmunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or V_(κ) domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides. Thelandscaping technique is not limited to producing antibodies comprisingketone moieties, but may be used instead to introduce a click chemistryreactive group, such as a nitrone, an azide or a cyclooctyne, onto anantibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of immunoconjugate formation are disclosed,for example, in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338;5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393;6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240,the Examples section of each incorporated herein by reference.

Other Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially with the subject ADCs. Alternatively, such agents may beconjugated to the antibodies of the invention, for example, drugs,toxins, oligonucleotides, immunomodulators, hormones, hormoneantagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesisinhibitors, etc. The therapeutic agents recited here are those agentsthat also are useful for administration separately with an ADC asdescribed above. Therapeutic agents include, for example, cytotoxicdrugs such as vinca alkaloids, anthracyclines such as doxorubicin,2-PDox or pro-2-PDox, gemcitabine, epipodophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, SN-38, COX-2inhibitors, antimitotics, anti-angiogenic and pro-apoptotic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinaseinhibitors, and others. Other useful anti-cancer cytotoxic drugs foradministering concurrently or sequentially, or for the preparation ofADCs include nitrogen mustards, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, COX-2 inhibitors, antimetabolites,pyrimidine analogs, purine analogs, platinum coordination complexes,mTOR inhibitors, tyrosine kinase inhibitors, proteosome inhibitors, HDACinhibitors, camptothecins, hormones, and the like. Suitable cytotoxicagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), as well as revised editions of these publications. Other suitablecytotoxic agents, such as experimental drugs, are known to those ofskill in the art. In a preferred embodiment, conjugates of camptothecinsand related compounds, such as SN-38, may be conjugated to hRS7 or otheranti-Trop-2 antibodies. In another preferred embodiment, gemcitabine isadministered to the subject in conjunction with SN-38-hRS7 and/or⁹⁰Y-hPAM4.

A toxin can be of animal, plant or microbial origin. Toxins of useinclude ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheriatoxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, forexample, Pastan et al., Cell 47:641 (1986), Goldenberg, C A—A CancerJournal for Clinicians 44:43 (1994), Sharkey and Goldenberg, C A—ACancer Journal for Clinicians 56:226 (2006). Additional toxins suitablefor use are known to those of skill in the art and are disclosed in U.S.Pat. No. 6,077,499, the Examples section of which is incorporated hereinby reference.

As used herein, the term “immunomodulator” includes a cytokine, alymphokine, a monokine, a stem cell growth factor, a lymphotoxin, ahematopoietic factor, a colony stimulating factor (CSF), an interferon(IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, a transforming growth factor (TGF), TGF-α, TGF-β,insulin-like growth factor (ILGF), erythropoietin, thrombopoietin, tumornecrosis factor (TNF), TNF-α, TNF-β, a mullerian-inhibiting substance,mouse gonadotropin-associated peptide, inhibin, activin, vascularendothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, Si factor, IL-1, IL-1cc, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18 IL-21 and IL-25, LIF, kit-ligand,FLT-3, angiostatin, thrombospondin, endostatin, lymphotoxin, and thelike.

Particularly useful therapeutic radionuclides include, but are notlimited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi ²¹³Bi, ²¹¹At, ⁶²CU, ⁶⁴CU, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹M, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹ Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.The therapeutic radionuclide preferably has a decay energy in the rangeof 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Augeremitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for analpha emitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Fm-255 and Th-227. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV.

For example, ⁹⁰Y, which emits an energetic beta particle, can be coupledto an antibody, antibody fragment or fusion protein, usingdiethylenetriaminepentaacetic acid (DTPA), or more preferably usingDOTA. Methods of conjugating ⁹⁰Y to antibodies or targetable constructsare known in the art and any such known methods may be used. (See, e.g.,U.S. Pat. No. 7,259,249, the Examples section of which is incorporatedherein by reference. See also Lindén et al., Clin Cancer Res.11:5215-22, 2005; Sharkey et al., J Nucl Med. 46:620-33, 2005; Sharkeyet al., J Nucl Med. 44:2000-18, 2003.)

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated antibody or antibody fragment. For example, theradiosensitizer can be used in combination with a radiolabeled antibodyor antibody fragment. The addition of the radiosensitizer can result inenhanced efficacy when compared to treatment with the radiolabeledantibody or antibody fragment alone. Radiosensitizers are described inD. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRCPress (1995). Other typical radionsensitizers of interest for use withthis technology include gemcitabine, 5-fluorouracil, and cisplatin, andhave been used in combination with external irradiation in the therapyof diverse cancers.

Antibodies or fragments thereof that have a boron addend-loaded carrierfor thermal neutron activation therapy will normally be affected insimilar ways. However, it will be advantageous to wait untilnon-targeted immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an anti-idiotypic antibodythat binds to the anti-cancer antibody. See U.S. Pat. No. 4,624,846 fora description of this general principle. For example, boron addends suchas carboranes, can be attached to antibodies. Carboranes can be preparedwith carboxyl functions on pendant side chains, as is well-known in theart. Attachment of carboranes to a carrier, such as aminodextran, can beachieved by activation of the carboxyl groups of the carboranes andcondensation with amines on the carrier. The intermediate conjugate isthen conjugated to the antibody. After administration of the antibodyconjugate, a boron addend is activated by thermal neutron irradiationand converted to radioactive atoms which decay by alpha-emission toproduce highly toxic, short-range effects.

Formulation and Administration

Suitable routes of administration of ADCs include, without limitation,oral, parenteral, rectal, transmucosal, intestinal administration,intramedullary, intrathecal, direct intraventricular, intravenous,intravitreal, intracavitary, intraperitoneal, or intratumoralinjections. The preferred routes of administration are parenteral, morepreferably intravenous. Alternatively, one may administer the compoundin a local rather than systemic manner, for example, via injection ofthe compound directly into a solid or hematological tumor.

ADCs can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the ADC is combined in amixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

In a preferred embodiment, the ADC is formulated in Good's biologicalbuffer (pH 6-7), using a buffer selected from the group consisting ofN-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The ADC can be formulated for intravenous administration via, forexample, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the ADC. For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of an ADC from sucha matrix depends upon the molecular weight of the ADC, the amount of ADCwithin the matrix, and the size of dispersed particles. Saltzman et al.,Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosageforms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

Generally, the dosage of an administered ADC for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. As discussedabove, dosages of antibody-SN-38 conjugates may vary from 3 to 18, morepreferably 4 to 16, more preferably 6 to 12, more preferably 8 to 10mg/kg. The dosage may be repeated as needed, for example, once per weekfor 2-10 weeks, once per week for 8 weeks, or once per week for 4 weeks.It may also be given less frequently, such as every other week forseveral months, or monthly or quarterly for many months, as needed in amaintenance therapy. The dosage is preferably administered multipletimes, once a week. A minimum dosage schedule of 4 weeks, morepreferably 8 weeks, more preferably 16 weeks or longer may be used, withthe dose frequency dependent on toxic side-effects and recoverytherefrom, mostly related to hematological toxicities. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 2, 4, 6, 8, 10, or 12 times or more.

Alternatively, an ADC may be administered as one dosage every 2 or 3weeks, repeated for a total of at least 3 dosages. Or, twice per weekfor 4-6 weeks. The dosage may be administered once every other week oreven less frequently, so the patient can recover from any drug-relatedtoxicities. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. The dosing schedule can optionally berepeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. In preferredembodiments, the method of the invention is used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

Kits

Various embodiments may concern kits containing components suitable fortreating cancer tissue in a patient. Exemplary kits may contain at leastone ADC as described herein. If the composition containing componentsfor administration is not formulated for delivery via the alimentarycanal, such as by oral delivery, a device capable of delivering the kitcomponents through some other route may be included. One type of device,for applications such as parenteral delivery, is a syringe that is usedto inject the composition into the body of a subject. Inhalation devicesmay also be used. In certain embodiments, an antibody or antigen bindingfragment thereof may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation of antibody (e.g., Kivitz et al., Clin. Ther.2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers.

In some embodiments, the containers may be vials that contain sterile,lyophilized formulations of a composition that are suitable forreconstitution. A kit may also contain one or more buffers suitable forreconstitution and/or dilution of other reagents. Other containers thatmay be used include, but are not limited to, a pouch, tray, box, tube,or the like. Kit components may be packaged and maintained sterilelywithin the containers. Another component that can be included isinstructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims.

Example 1 Targeted Therapy of GI Cancers with IMMU-132 (IsactuzumabGovitecan), an Anti-Trop-2-SN-38 Antibody Drug Conjugate (ADC)

Trop-2 is a tumor-associated glycoprotein highly prevalent in manyepithelial cancers. Its elevated expression has been linked to moreaggressive disease and a poor prognosis. A humanized mAb binding to theextracellular domain of Trop-2 was conjugated to SN-38 (IMMU-132;average drug:mAb ratio=7.6), the active principle of CPT-11. Afterpotent activity in human tumor xenografts, a Phase I/II trial wasinitiated in patients (pts) with diverse solid tumors, including GIcancers.

Methods:

Patients with metastatic cancers were enrolled after failing standardtherapy, starting at a dose of 8.0 mg/kg given on days 1 and 8 of a3-week cycle. The MTD was determined to be 12 mg/kg; dose levels of 8and 10 mg/kg were chosen for Phase II testing.

Results:

Sixty patients with advanced GI cancers were enrolled in a Phase I/IItrial. In 29 CRC patients (9 treated at 10 mg/kg, 20 at 8 mg/kg), 1 hada PR (partial response) and 14 had SD (stable disease) as the bestresponse by RECIST, with a time to progression (TTP) of 50+ wks for thePR (−65%) and a median of 21+ wks for the SD patients (5 continuing).Thirteen CRC patients had KRAS mutations, 7 showing SD with a median TTPof 19.1+ wks (range, 12.0-34.0; 3 continuing). Of 15 pancreatic cancerpatients that were treated (5 at 8, 7 at 10, and 3 at 12 mg/kg), 7 hadSD as best response for a median TTP of 15.0 wks. Among 11 patients withesophageal cancer (9 started at 8, 1 at 10, and 1 at 18 mg/kg), 8 had CTassessment, showing 1 PR with a TTP of 30+ wks, and 4 with SD of 17.4+,21.9, 26.3, and 29.9 wks. Of 5 gastric cancer patients (2 at 8 and 3 at10 mg/kg), only 3 have had CT assessment, all with SD (1 with 19% targetlesion reduction and an ongoing TTP of 29+ wks).

Neutropenia was the principal dose-limiting toxicity, with fatigue,diarrhea, nausea, and vomiting as other commonly reported toxicities.However, the toxicity profile from 75 patients in the full trial showedonly 17.3% and 2.7% Grade 3 and Grade 4 neutropenia, respectively, andjust 6.7% Grade 3 diarrhea.

Conclusions:

IMMU-132 showed a high therapeutic index in patients with diverserelapsed metastatic GI cancers. It has a moderately-toxic drugconjugated to an internalizing, cancer-selective mAb, which can be givenrepeatedly over many months once weekly×2 in a 21-day cycle.

Example 2 Anti-CEACAM5-SN-38 Antibody Drug Conjugate (IMMU-130) Activityin Metastatic Colorectal Cancer (mCRC)

IMMU-130 is a CEACAM5-targeted ADC, labetuzumab-SN-38, with the drugbeing the active form of the topoisomerase I inhibitor, CPT-11, andsubstituted at 7-8 moles/mole of IgG. This agent is in Phase I/IIclinical trials in patients with relapsed mCRC.

Methods:

Experiments were conducted in female athymic nude mice, 4 weeks orolder, bearing s.c. LS 174T human colon carcinoma xenografts of (˜0.2cm³ size), or 2 weeks after lung metastases were generated by i.v.injection of GW-39 human colon carcinoma cells. Untreated controls,including a non-targeting ADC, were included. Biodistribution wasexamined in the s.c model using single 12.5 mg/kg dose of the ADC orunconjugated labetuzumab, each spiked with ¹¹¹In-labeled substrate.Tolerability studies were conducted in white New Zealand rabbits.

Results:

In the metastatic model (n=8), fractionated dosing of 2 cycles of a21-day cycle therapy, with a fixed total dose of 50 mg/kg of ADC, showedthat twice-weekly×2 weeks and once weekly×2 weeks schedules doubledmedian survival vs. untreated mice, and was better than the once for 2weeks schedule (P<0.0474; log-rank). Pre-dosing with as much as twicethe dose of labetuzumab as the ADC dose, in the metastatic model (n=10),did not affect median survival (P>0.15). Therapy experiments in the s.c.model revealed that the linker in IMMU-130, liberating 50% of drug in˜20 h, was superior to the conjugate with an ultrastable linker (n=5),that the ADC was better than an MTD of 5FU/leucovorin chemotherapy(n=10; P<0.0001), and that the ADC could be combined with bevacizumabfor improved efficacy (n=8-10; P<0.031). Significantly better efficacyfor the specific ADC vs. nonspecific ADC was observed. Pharmacokineticsin mice indicated˜25% longer half-life for MAb vs. ADC, but with minimalimpact on tumor uptake. A tolerability study in rabbits showed the NOAELto be the human equivalent dose of 40-60 mg/kg, given as two doses.

Conclusions:

Preclinical data showed an excellent therapeutic window for this ADC,which appears to be translated into the clinical experience thus far.The potential for combination therapy is also indicated.

Example 3 Production and Use of Anti-Trop-2-SN-38 Antibody-DrugConjugate

The humanized RS7 (hRS7) anti-Trop-2 antibody was produced as describedin U.S. Pat. No. 7,238,785, the Figures and Examples section of whichare incorporated herein by reference. SN-38 attached to a CL2A linkerwas produced and conjugated to hRS7 (anti-Trop-2), hPAM4 (anti-MUC5ac),hA20 (anti-CD20) or hMN-14 (anti-CEACAM5) antibodies according to U.S.Pat. No. 7,999,083 (Example 10 and 12 of which are incorporated hereinby reference). The conjugation protocol resulted in a ratio of about 6SN-38 molecules attached per antibody molecule.

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4(anti-MUC-5ac), and hMN-14 (anti-CEACAM5) antibodies showed betterefficacies than control hA20-CL2A-SN-38 conjugate (anti-CD20) anduntreated control. Similarly in a BXPC3 model of human pancreaticcancer, the specific hRS7-CL2A-SN-38 showed better therapeutic efficacythan control treatments (FIG. 2).

Example 4 Efficacy of Anti-Trop-2 Antibody Conjugated to a Prodrug Formof 2-Pyrrolinodoxorubicin (2-PDox)

A prodrug form of 2-PDox (referred to as pro-2-PDox) was prepared andconjugated to antibodies as disclosed in U.S. patent application Ser.No. 14/175,089 (Example 1 of which is incorporated herein by reference).The structures of doxorubicin, 2-PDox, pro-2-PDox and a maleimideactivated form of pro-2-PDox that is suitable for conjugation tosulfhydryl groups on antibodies or other proteins are shown in FIG. 3.Unless otherwise stated below, the number of drug moieties per antibodymolecule was in the range of about 6.5 to about 7.5.

In Vitro Cell-Binding Studies—

Retention of antibody binding was confirmed by cell binding assayscomparing binding of the conjugated to the unconjugated antibody (Chari,2008, Acc Chem Res 41:98-107). The potency of the conjugate was testedin a 4-day MTS assay using appropriate target cells. The anti-Trop-2 ADC(hRS7-pro-2-PDox) exhibited IC₅₀ values of 0.35-1.09 nM in gastric(NCI-N87), pancreatic (Capan-1), and breast (MDA-MB-468) human cancercell lines, with free drug exhibiting 0.02-0.07 nM potency in the samecell lines. In additional studies, hRS7-pro-2-PDox was observed to becytotoxic to MDA-MB-468, AG S, NCI-N87 and Capan-1 solid tumor celllines (not shown).

No significant difference in binding of the antibody moiety to NCI-N87gastric carcinoma cells was observed between unconjugated hRS7 andpro-2-PDox-hRS7 conjugated to 6 molecules of pro-2-PDox per antibody(not shown). It is concluded that conjugation of pro-2-PDox toantibodies does not affect antibody-antigen binding activity.

Serum Stability—

Serum stability of anti-Trop-2 ADC (hRS7-pro-2-PDox) was determined byincubation in human serum at a concentration of 0.2 mg/mL at 37° C. Theincubate was analyzed by HPLC using butyl hydrophobic interactionchromatography (HIC). The analysis showed that there was no release offree drug from the conjugate, suggesting high serum stability of theconjugate. When the same experiment was repeated with hRS7-doxorubicinconjugate, containing the same cleavable linker as hRS7-pro-2-PDox, andwhere the free drug was independently verified to be released with ahalf-life of 96 h, clear formation of a peak corresponding to freedoxorubicin was seen on HIC HPLC.

Surprisingly, it was determined that the pro-2-PDox conjugate was heldtightly to the antibody because it cross-linked the peptide chains ofthe antibody together. The cross-linking stabilizes the attachment ofthe drug to the antibody so that the drug is only releasedintracellularly after the antibody is metabolized. The cross-linkingassists in minimizing toxicity, for example cardiotoxicity, that wouldresult from release of free drug in circulation. Previous use of 2-PDoxpeptide conjugates failed because the drug cross-linked the peptide toother proteins or peptides in vivo. With the present anti-Trop-2 ADC,the pro-2-PDox is attached to interchain disulfide thiol groups while inthe prodrug form. The prodrug protection is rapidly removed in vivo soonafter injection and the resulting 2-PDox portion of the conjugatecross-links the peptide chains of the antibody, forming intramolecularcross-linking within the antibody molecule. This both stabilizes the ADCand prevents cross-linking to other molecules in circulation.

In Vivo Preclinical Studies—

Tumor size was determined by caliper measurements of length (L) andwidth (W) with tumor volume calculated as (L×W²)/2. Tumors were measuredand mice weighed twice a week. Mice were euthanized if their tumorsreached >1 cm³ in size, lost greater than 15% of their starting bodyweight, or otherwise became moribund. Statistical analysis for the tumorgrowth data was based on area under the curve (AUC) and survival time.Profiles of individual tumor growth were obtained through linear curvemodeling. An f-test was employed to determine equality of variancebetween groups prior to statistical analysis of growth curves. Atwo-tailed t-test was used to assess statistical significance betweenall the various treatment groups and non-specific controls. For thesaline control analysis a one-tailed t-test was used to assesssignificance. Survival studies were analyzed using Kaplan-Meier plots(log-rank analysis), using the Prism GraphPad Software (v4.03) softwarepackage (Advanced Graphics Software, Inc.; Encinitas, Calif.). All dosesin preclinical experiments are expressed in antibody amounts. In termsof drug, 100 μg of antibody (5 mg/kg) in a 20-g mouse, for example,carries 1.4 μg-2.8 μg (0.14-0.17 mg/kg) of pro-2-PDox equivalent dosewhen using an ADC with 3-6 drugs/IgG.

A single i.v. dose of ≧300 μg [˜10 μg of pro-2-PDox] of the anti-Trop-2ADC was lethal, but 4 doses of 45 μg given in 2 weeks were tolerated byall animals. Using this dosing regimen, we examined the therapeuticeffect of anti-Trop-2 hRS7-pro-2-PDox in 2 human tumor xenograft models,Capan-1 (pancreatic cancer) and NCI-N87 (gastric cancer). Therapy began7 days after tumor transplantation in nude mice. In the established,7-day-old, Capan-1 model, 100% of established tumors quickly regressed,with no evidence of re-growth (FIG. 4). This result was reproduced in arepeat experiment (not shown). The anti-Trop-2 conjugate of pro-2-PDoxwas much more effective than the same drug conjugated to an antibody(hMN-14) against CEACAM5, which is also expressed in pancreatic cancer,or an antibody against CD20 (hA20), which is not. All treatments weresuperior to the saline control.

Similar results were observed in the established NCI-N87 model (FIG.5A), where a 2^(nd) course of therapy, administered after day 70, wassafely tolerated and led to further regressions of residual tumor (FIG.5A). The internalizing hRS7-SN-38 conjugate, targeting Trop-2, providedbetter therapeutic responses than a conjugate of a poorly internalizinganti-CEACAM5 antibody, hMN-14 (FIG. 4, FIG. 5). A non-targeted anti-CD20ADC, hA20-pro-2-PDox, was ineffective, indicating selective therapeuticefficacy (FIG. 4, FIG. 5). Data from a breast cancer xenograft(MDA-MB-468) and a second pancreatic cancer xenograft (FIG. 5B and FIG.5C, respectively) showed the same pattern, with the anti-Trop-2 ADCsignificantly more efficacious compared to non-targeting ADC or salinecontrol. In both cases, administration of anti-Trop-2 ADC produced aclear inhibition of tumor growth to the end of the study.

PK and Toxicity of hRS7-Pro-2-PDox with Substitutions of 6.8 or 3.7Drug/IgG—

Antibody-drug conjugates (ADCs) carrying as much as 8 ultratoxicdrugs/MAb are known to clear faster than unmodified MAb and to increaseoff-target toxicity, a finding that has led to the current trends to usedrug substitutions of ≦4 (Hamblett et al., 2004, Clin Cancer Res10:7063-70). ADCs were prepared and evaluated with mean drug/MAbsubstitution ratios (MSRs) of ˜6:1 and ˜3:1. Groups of normal mice (n=5)were administered, i.v., single doses of unmodified hRS7 orhRS7-pro-2-PDox with drug substitution of 6.8 or 3.7 (same proteindose), and serum samples were collected at 30 min, 4 h, 24 h, 72 h, and168 h post-injection. These were analyzed by ELISA for antibodyconcentration. There were no significant differences in serumconcentrations at various times, indicating that these showed similarclearance from the blood. The PK parameters (Cmax, AUC, etc.) were alsosimilar. ADCs with either higher or lower drug substitution had similartolerability in nude mice, when the administered at the same dose ofconjugated drug.

Therapeutic Efficacy at Minimum Effective Dose (MED)—

Anti-Trop-2 ADC (hRS7-pro-2-PDox), was evaluated in nude mice bearingNCI-N87 human gastric cancer xenografts by administering a single bolusprotein dose of 9 mg/kg, 6.75 mg/kg, 4.5 mg/kg, 2.25 mg/kg, or 1 mg/kg.The therapy was started when the mean tumor volume (mTV) was 0.256 cm³.On day 21, mTV in the saline control group (non-treatment group) was0.801±0.181 cm³ which was significantly larger than that in mice treatedwith 9, 6.75, 4.5, or 2.25 mg/kg dose with mTV of 0.211±0.042 cm³,0.239±0.0.054 cm³, 0.264±0.087 cm³, and 0.567±0.179 cm³, respectively(P<0.0047, one tailed t-test). From these, the minimum effective dosewas estimated to be 2.25 mg/kg, while 9 mg/kg represented MTD.

Example 5 Additional Studies with Anti-Trop-2 Pro-2-PDox ADC

Further in vivo efficacy studies were performed in nude mice implantedwith NCI-N87 human gastric cancer xenografts (FIG. 6A-F). One treatmentcycle with 4×45 μg of hRS7-pro-2-PDox rapidly regressed all tumors (FIG.6D). A second treatment cycle was initiated about 2 months after the endof the first cycle, resulting in complete regression of all but one ofthe hRS7-pro-2-PDox treated animals. The hA20 (anti-CD20), hLL1(anti-CD22) and hMN-14 (anti-CEACAM5) conjugates had little effect ontumor progression (FIGS. 6B, 6E and 6F) compared to saline control (FIG.6A). Administration of pro-2-PDox-hMN-15 (anti-CEACAM6) resulted in adelayed regression of gastric cancer (FIG. 6C), which was less effectivethan the hRS7 conjugate (FIG. 6D).

The effect of varying dosage schedule of anti-Trop-2 ADC on anti-tumorefficacy was examined (FIG. 7, FIG. 8A-G). The experiment began 9 daysafter tumor implantation when mean tumor volume for all groups was 0.383cm³, and ended on day 93 (84 days after initiation of therapy). In thisstudy, administration of anti-Trop-2 ADC as a single dose of 180 μg, twoweekly doses of 90 μg, and q4d×4 of 45 μg all resulted in significantlyenhanced survival (FIG. 7, FIG. 8B-D). For the saline control, 0 of 9mice survived (FIG. 8A). For mice receiving 45 μg q4d×4 ofhRS7-pro-2-PDox, 8 of 9 mice were alive at day 94 (FIG. 8B). For micereceiving 90 μg weekly×2 of hRS7-pro-2-PDox, 9 of 9 mice were alive atday 94 (FIG. 8C). For mice receiving a single dose of 180 μg ofhRS7-pro-2-PDox, 7 of 9 mice were alive at day 94 (FIG. 8D).

At the same dosage schedule, the control hA20 conjugate had no effect onsurvival (FIG. 7, FIG. 8E-F). A toxicity study showed that the threedosage schedules of hRS7-pro-2-PDox resulted in similarly low levels oftoxicity (not shown).

The hRS7-pro-2-PDox conjugate was also effective in Capan-1 pancreaticcancer (not shown) and was more effective at inhibiting tumor growththan a hRS7-SN-38 conjugate (not shown). The hPAM4-pro-2-PDox conjugatewas also more effective at inhibiting growth of Capan-1 human pancreaticcancer than an hPAM4-SN-38 conjugate (not shown). At 63 days afterCapan-1 tumor injection (with therapy starting at 1 dayspost-innoculation), 0 of 10 mice were alive in the saline control, 10 of10 mice were alive in mice treated twice weekly×2 weeks with 45 μg ofhPAM4-pro-2-PDox, 2 of 10 mice were alive in mice treated twice weekly×2weeks with 45 μg of hA20-pro-2-PDox, 0 of 10 mice were alive in micetreated twice weekly×4 weeks with 250 μg of hPAM4-SN-38, and 0 of 10mice were alive in mice treated twice weekly×4 weeks with 250 μg ofh20-SN-38.

hRS7-pro-2-PDox was substantially more effective than hRS7-SN-38 atinhibiting growth of PxPC-3 pancreatic cancer (not shown) and wasslightly more effective than hRS7-SN-38 at inhibiting growth ofMDA-MB-468 breast cancer (not shown).

The effect of different single doses of hRS7-pro-2-PDox on growth ofNCI-N87 gastric carcinoma xenografts is shown in FIG. 9. Using a singledose, the maximum effect on tumor growth was observed at 90 μg or higher(FIG. 9).

Survival curves for mice bearing NCI-N87 human gastric carcinomaxenografts and administered a single dose of anti-Trop-2 ADC are shownin FIG. 10. A single dose of 45 μg was the minimum required to see asignificant survival benefit compared to saline control (FIG. 10). Miceadministered single doses of 90 μg or higher showed 100% survival to thetermination of the experiment.

The ADCC activity of various hRS7-ADC conjugates was determined incomparison to hRS7 IgG (FIG. 11). PBMCs were purified from bloodpurchased from the Blood Center of New Jersey. A Trop-2-positive humanpancreatic adenocarcinoma cell line (BxPC-3) was used as the target cellline with an effector to target ratio of 100:1. ADCC mediated by hRS7IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reducedand capped hRS7-NEM. All were used at 33.3 nM.

Results are shown in FIG. 11. Overall activity was low, but significant.There was 8.5% specific lysis for the hRS7 IgG which was notsignificantly different from hRS7-Pro-2-PDox. Both were significantlybetter than hLL2 control and hRS7-NEM and hRS7-SN-38 (P<0.02, two-tailedt-test). There was no difference between hRS7-NEM and hRS7-SN-38.

Example 6 Efficacy of Anti-Trop-2-SN-38 ADC Against Diverse EpithelialCancers In Vivo

Abstract

The purpose of this study was to evaluate the efficacy of anSN-38-anti-Trop-2 (hRS7) ADC against several human solid tumor types,and to assess its tolerability in mice and monkeys, the latter withtissue cross-reactivity to hRS7 similar to humans. Two SN-38derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-Trop-2-humanized antibody, hRS7. The immunoconjugates werecharacterized in vitro for stability, binding, and cytotoxicity.Efficacy was tested in five different human solid tumor-xenograft modelsthat expressed Trop-2 antigen. Toxicity was assessed in mice and inCynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≦0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

In summary, the anti-Trop-2 hRS7-CL2A-SN-38 ADC provided significant andspecific antitumor effects against a range of human solid tumor types.It was well tolerated in monkeys, with tissue Trop-2 expression similarto humans, at clinically relevant doses.

Introduction

Successful irinotecan treatment of patients with solid tumors has beenlimited, due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-Trop-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., ≦3.2 mg/kg cumulative SN-38 equivalent dose). Trop-2 is widelyexpressed in many epithelial cancers, but also some normal tissues, andtherefore a dose escalation study in Cynomolgus monkeys was performed toassess the clinical safety of this conjugate. Monkeys tolerated 24 mgSN-38 equivalents/kg with only minor, reversible, toxicities. Given itstumor-targeting and safety profile, hRS7-SN-38 provides a significantimprovement in the management of solid tumors responsive to irinotecan.

Material and Methods

Cell Lines, Antibodies, and Chemotherapeutics—

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection. These include Calu-3 (non-small celllung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205(colonic adenocarcinoma), Capan-1 and BxPC-3 (pancreaticadenocarcinomas), and PC-3 (prostatic adenocarcinomas). Humanized RS7IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22(hLL2 IgG, epratuzumab) antibodies were prepared at Immunomedics, Inc.Irinotecan (20 mg/mL) was obtained from Hospira, Inc.

SN-38 Immunoconjugates and In Vitro Aspects—

Synthesis of CL2-SN-38 has been described previously (Moon et al., 2008,J Med Chem 51:6916-26). Its conjugation to hRS7 IgG and serum stabilitywere performed as described (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Chem Res 15:6052-61). Preparations ofCL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, and stability, binding,and cytotoxicity studies, were conducted as described in the precedingExamples.

In Vivo Therapeutic Studies—

For all animal studies, the doses of SN-38 immunoconjugates andirinotecan are shown in SN-38 equivalents. Based on a mean SN-38/IgGsubstitution ratio of 6, a dose of 500 μg ADC to a 20-g mouse (25 mg/kg)contains 0.4 mg/kg of SN-38. Irinotecan doses are likewise shown asSN-38 equivalents (i.e., 40 mg irinotecan/kg is equivalent to 24 mg/kgof SN-38).

NCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, and maleSwiss-Webster mice, 10 weeks old, were purchased from Taconic Farms.Tolerability studies were performed in Cynomolgus monkeys(Macacafascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd.

Animals were implanted subcutaneously with different human cancer celllines. Tumor volume (TV) was determined by measurements in 2 dimensionsusing calipers, with volumes defined as: L× w²/2, where L is the longestdimension of the tumor and w is the shortest. Tumors ranged in sizebetween 0.10 and 0.47 cm³ when therapy began. Treatment regimens,dosages, and number of animals in each experiment are described in theResults. The lyophilized hRS7-CL2A-SN-38 and control ADC werereconstituted and diluted as required in sterile saline. All reagentswere administered intraperitoneally (0.1 mL), except irinotecan, whichwas administered intravenously. The dosing regimen was influenced by ourprior investigations, where the ADC was given every 4 days or twiceweekly for varying lengths of time (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). Thisdosing frequency reflected a consideration of the conjugate's serumhalf-life in vitro, to allow a more continuous exposure to the ADC.

Statistics—

Growth curves are shown as percent change in initial TV over time.Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups before statistical analysis of growth curves. A2-tailed t-test was used to assess statistical significance between thevarious treatment groups and controls, except for the saline control,where a 1-tailed t-test was used (significance at P≦0.05). Statisticalcomparisons of AUC were performed only up to the time that the firstanimal within a group was euthanized due to progression.

Pharmacokinetics and Biodistribution—

¹¹¹n-radiolabeled hRS7-CL2A-SN-38 and hRS7 IgG were injected into nudemice bearing s.c. SK-MES-1 tumors (˜0.3 cm³). One group was injectedintravenously with 20 μCi (250-μg protein) of ¹¹¹In-hRS7-CL2A-SN-38,whereas another group received 20 μCi (250-g protein) of ¹¹¹In-hRS7 IgG.At various timepoints mice (5 per timepoint) were anesthetized, bled viaintracardiac puncture, and then euthanized. Tumors and various tissueswere removed, weighed, and counted by γ scintillation to determine thepercentage injected dose per gram tissue (% ID/g). A third group wasinjected with 250 μg of unlabeled hRS7-CL2A-SN-38 3 days before theadministration of ¹¹¹In-hRS7-CL2A-SN-38 and likewise necropsied. A2-tailed t-test was used to compare hRS7-CL2A-SN-38 and hRS7 IgG uptakeafter determining equality of variance using the f-test. Pharmacokineticanalysis on blood clearance was performed using WinNonLin software(Parsight Corp.).

Tolerability in Swiss-Webster Mice and Cynomolgus Monkeys—

Briefly, mice were sorted into 4 groups each to receive 2-mL i.p.injections of either a sodium acetate buffer control or 3 differentdoses of hRS7-CL2A-SN-38 (4, 8, or 12 mg/kg of SN-38) on days 0 and 3followed by blood and serum collection, as described in Results.Cynomolgus monkeys (3 male and 3 female; 2.5-4.0 kg) were administered 2different doses of hRS7-CL2A-SN-38. Dosages, times, and number ofmonkeys bled for evaluation of possible hematologic toxicities and serumchemistries are described in the Results.

Results

Stability and Potency of hRS7-CL2A-SN-38—

Two different linkages were used to conjugate SN-38 to hRS7 IgG (FIG.12A). The first is termed CL2-SN-38 and has been described previously(Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, ClinChem Res 15:6052-61). A change in the synthesis of CL2 to remove thephenylalanine moiety within the linker was used to produce the CL2Alinker. This change simplified the synthesis, but did not affect theconjugation outcome (e.g., both CL2-SN-38 and CL2A-SN-38 incorporated ˜6SN-38 per IgG molecule). Side-by-side comparisons found no significantdifferences in serum stability, antigen binding, or in vitrocytotoxicity. This result was surprising, since the phenylalanineresidue in CL2 is part of a designed cleavage site for cathepsin B, alysosomal protease.

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 (FIG. 12B) or Capan-1 tumors (FIG. 12C), using 0.4mg or 0.2 mg/kg SN-38 twice weekly×4 weeks, respectively, and withstarting tumors of 0.25 cm³ size in both studies. Both the hRS7-CL2A andCL2-SN-38 conjugates significantly inhibited tumor growth compared tountreated (AUC_(14days) P<0.002 vs. saline in COLO 205 model;AUC_(21days) P<0.001 vs. saline in Capan-1 model), and a nontargetinganti-CD20 control ADC, hA20-CL2A-SN-38 (AUC_(14days) P<0.003 in COLO-205model; AUC_(35days): P<0.002 in Capan-1 model). At the end of the study(day 140) in the Capan-1 model, 50% of the mice treated withhRS7-CL2A-SN-38 and 40% of the hRS7-CL2-SN-38 mice were tumor-free,whereas only 20% of the hA20-ADC-treated animals had no visible sign ofdisease. As demonstrated in FIG. 12, the CL2A linker resulted in asomewhat higher efficacy compared to CL2.

Mechanism of Action—

In vitro cytotoxicity studies demonstrated that hRS7-CL2A-SN-38 had IC₅₀values in the nmol/L range against several different solid tumor lines(Table 6). The IC₅₀ with free SN-38 was lower than the conjugate in allcell lines. Although there was no apparent correlation between Trop-2expression and sensitivity to hRS7-CL2A-SN-38, the IC₅₀ ratio of the ADCversus free SN-38 was lower in the higher Trop-2-expressing cells, mostlikely reflecting the enhanced ability to internalize the drug when moreantigen is present. SN-38 is known to activate several signalingpathways in cells, leading to apoptosis (e.g., Cusack et al., 2001,Cancer Res 61:3535-40; Liu et al. 2009, Cancer Lett 274:47-53; Lagadecet al., 2008, Br J Cancer 98:335-44). Our initial studies examined theexpression of 2 proteins involved in early signaling events(p21^(Waf1/Cip1) and p53) and 1 late apoptotic event [cleavage ofpoly-ADP-ribose polymerase (PARP)] in vitro (not shown). In BxPC-3,SN-38 led to a 20-fold increase in p21 ^(Waf1/Cip1) expression (notshown), whereas hRS7-CL2A-SN-38 resulted in only a 10-fold increase (notshown), a finding consistent with the higher activity with free SN-38 inthis cell line (Table 6). However, hRS7-CL2A-SN-38 increasedp21^(Waf1/Cip1) expression in Calu-3 more than 2-fold over free SN-38(not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression (not shown). In bothBxPC-3 and Calu-3, upregulation of p53 with free SN-38 was not evidentuntil 48 hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours(not shown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure (not shown). In terms of laterapoptotic events, cleavage of PARP was evident in both cell lines whenincubated with either SN-38 or the conjugate (not shown). The presenceof the cleaved PARP was higher at 24 hours in BxPC-3 (not shown), whichcorrelates with high expression of p21 and its lower IC₅₀. The higherdegree of cleavage with free SN-38 over the ADC was consistent with thecytotoxicity findings.

Efficacy of hRS7-SN-38—

Because Trop-2 is widely expressed in several human carcinomas, studieswere performed in several different human cancer models, which startedusing the hRS7-CL2-SN-38 linkage, but later, conjugates with theCL2A-linkage were used. Calu-3-bearing nude mice given 0.04 mg SN-38/kgof the hRS7-CL2-SN-38 every 4 days×4 had a significantly improvedresponse compared to animals administered the equivalent amount ofnon-targeting hLL2-CL2-SN-38 (TV=0.14±0.22 cm³ vs. 0.80±0.91 cm³,respectively; AUC_(42days) P<0.026; FIG. 13A). A dose-response wasobserved when the dose was increased to 0.4 mg/kg SN-38 (FIG. 13A). Atthis higher dose level, all mice given the specific hRS7 conjugate were“cured” within 28 days, and remained tumor-free until the end of thestudy on day 147, whereas tumors regrew in animals treated with theirrelevant ADC (specific vs. irrelevant AUC_(98days): P=0.05). In micereceiving the mixture of hRS7 IgG and SN-38, tumors progressed >4.5-foldby day 56 (TV=1.10±0.88 cm³; AUC_(56days) P<0.006 vs. hRS7-CL2-SN-38)(FIG. 13A).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG. 13Bhttp://clincancerres.aacriournals.org/content/17/10/3157.long-F3),hRS7-CL2-SN-38 (0.4 mg/kg, q4d×8) prevented tumor growth over the 28-daytreatment period with significantly smaller tumors compared to controlanti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³, 1.19±0.59cm³, and 1.77±0.93 cm³, respectively; AUC_(28days) P<0.016).

TABLE 6 Expression of Trop-2 in vitro cytotoxicity of SN-38 andhRS7-SN-38 in various solid tumor lines Trop-2 expression via FACSCytotoxicity results Median SN-38 95% CI hRS7-SN-38 95% CI ADC/freefluorescence Percent IC₅₀ IC₅₀ IC₅₀ IC₅₀ SN-38 Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) ratio Calu-3 282.2 (4.7)99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 205 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95 1.26-3.01 1.91 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99 5.02-9.72 2.00 PC-3  46.2 (5.5) 73.6% 1.86 1.16-2.99 4.242.99-6.01 2.28 SK-MES-1  44.0 (3.5) 91.2% 8.61  6.30-11.76 23.1417.98-29.78 2.69 BxPC-3  26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03 3.25-4.982.80

The MTD of irinotecan (24 mg SN-38/kg, q2d×5) was as effective ashRS7-CL2-SN-38 in COLO 205 cells, because mouse serum can moreefficiently convert irinotecan to SN-38 (Morton et al., 2000, Cancer Res60:4206-10) than human serum, but the SN-38 dose in irinotecan (2,400 μgcumulative) was 37.5-fold greater than with the conjugate (64 μg total).

Animals bearing Capan-1 (FIG. 13C) showed no significant response toirinotecan alone when given at an SN-38-dose equivalent to thehRS7-CL2-SN-38 conjugate (e.g., on day 35, average tumor size was0.04±0.05 cm³ in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62cm³ in irinotecan-treated animals given 0.4 mg/kg SN-38;AUC_(day35)P<0.001; FIG. 13C). When the irinotecan dose was increased10-fold to 4 mg/kg SN-38, the response improved, but still was not assignificant as the conjugate at the 0.4 mg/kg SN-38 dose level(TV=0.17±0.18 cm³ vs. 1.69±0.47 cm³, AUC_(day49)P<0.001) (FIG. 13C). Anequal dose of nontargeting hA20-CL2-SN-38 also had a significantantitumor effect as compared to irinotecan-treated animals, but thespecific hRS7 conjugate was significantly better than the irrelevant ADC(TV=0.17±0.18 cm³ vs. 0.80±0.68 cm³, AUC_(day49)P<0.018) (FIG. 13C).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 13D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively;AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38equivalent dose (TV=0.27-0.18 cm³ vs. 0.90±0.62 cm³, respectively;AUC_(day25)P<0.004) (FIG. 13D). Interestingly, in mice bearing SK-MES-1human squamous cell lung tumors treated with 0.4 mg/kg of the ADC (FIG.13E), tumor growth inhibition was superior to saline or unconjugatedhRS7 IgG (TV=0.36±0.25 cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³,respectively; AUC₂d., P<0.043), but nontargeting hA20-CL2A-SN-38 or theMTD of irinotecan provided the same antitumor effects as the specifichRS7-SN-38 conjugate (FIG. 13E).

In all murine studies, the hRS7-SN-38 ADC was well tolerated in terms ofbody weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38—

The biodistributions of hRS7-CL2A-SN-38 or unconjugated hRS7 IgG werecompared in mice bearing SK-MES-1 human squamous cell lung carcinomaxenografts (not shown), using the respective ¹¹¹In-labeled substrates. Apharmacokinetic analysis was performed to determine the clearance ofhRS7-CL2A-SN-38 relative to unconjugated hRS7 (not shown). The ADCcleared faster than the equivalent amount of unconjugated hRS7, with theADC exhibiting ˜40% shorter half-life and mean residence time.Nonetheless, this had a minimal impact on tumor uptake (not shown).Although there were significant differences at the 24- and 48-hourtimepoints, by 72 hours (peak uptake) the amounts of both agents in thetumor were similar. Among the normal tissues, hepatic and splenicdifferences were the most striking (not shown). At 24 hourspostinjection, there was >2-fold more hRS7-CL2A-SN-38 in the liver thanhRS7 IgG (not shown). Conversely, in the spleen there was 3-fold moreparental hRS7 IgG present at peak uptake (48-hour timepoint) thanhRS7-CL2A-SN-38 (not shown). Uptake and clearance in the rest of thetissues generally reflected differences in the blood concentration (notshown).

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123; notshown http://clincancerres.aacrjournals.org/content/17/10/3157.long-F4).Predosing had no appreciable impact on blood clearance or tissue uptake(not shown). These studies suggest that in some tumor models, tumoraccretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster Mice and CynomolgusMonkeys

Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8, and 12mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weight loss(not shown). No hematopoietic toxicity occurred and serum chemistriesonly revealed elevated aspartate transaminase (AST, FIG. 14A) andalanine transaminase (ALT, FIG. 14B). Seven days after treatment, ASTrose above normal levels (>298 U/L) in all 3 treatment groups (FIG.14A), with the largest proportion of mice being in the 2×8 mg/kg group.However, by 15 days posttreatment, most animals were within the normalrange. ALT levels were also above the normal range (>77 U/L) within 7days of treatment (FIG. 14B) and with evidence of normalization by Day15. Livers from all these mice did not show histologic evidence oftissue damage (not shown). In terms of renal function, only glucose andchloride levels were somewhat elevated in the treated groups. At 2×8mg/kg, 5 of 7 mice had slightly elevated glucose levels (range of273-320 mg/dL, upper end of normal 263 mg/dL) that returned to normal by15 days postinjection. Similarly, chloride levels were slightlyelevated, ranging from 116 to 127 mmol/L (upper end of normal range 115mmol/L) in the 2 highest dosage groups (57% in the 2×8 mg/kg group and100% of the mice in the 2×12 mg/kg group), and remained elevated out to15 days postinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express Trop-2 identified by hRS7, a more suitablemodel was required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (neutrophil and platelet data shown in FIG. 14Cand FIG. 14D), but values did not fall below normal ranges. No abnormalvalues were found in the serum chemistries. Histopathology of theanimals necropsied on day 11 (8 days after last injection) showedmicroscopic changes in hematopoietic organs (thymus, mandibular andmesenteric lymph nodes, spleen, and bone marrow), gastrointestinalorgans (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum),female reproductive organs (ovary, uterus, and vagina), and at theinjection site. These changes ranged from minimal to moderate and werefully reversed at the end of the recovery period (day 32) in alltissues, except in the thymus and gastrointestinal tract, which weretrending towards full recovery at this later timepoint (not shown).

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group (not shown). These dataindicate that dose-limiting toxicities were identical to that ofirinotecan; namely, intestinal and hematologic. Thus, the MTD forhRS7-CL2A-SN-38 lies between 2×0.96 and 1.92 mg SN-38/kg, whichrepresents a human equivalent dose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

Trop-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents (Ohmachi etal., 2006, Clin Cancer Res 12:3057-63; Fong et al., 2008, Br J Cancer99:1290-95; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14). TheRS7 antibody internalizes when bound to Trop-2 (Shih et al., 1995,Cancer Res 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38(Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. To simplify the synthetic process, inCL2A the phenylalanine was eliminated, and thus the cathepsin B cleavagesite was removed. Interestingly, this product had a better-definedchromatographic profile compared to the broad profile obtained with CL2(not shown), but more importantly, this change had no impact on theconjugate's binding, stability, or potency in side-by-side testing.These data suggest that SN-38 in CL2 was released from the conjugateprimarily by the cleavage at the pH-sensitive benzyl carbonate bond toSN-38's lactone ring and not the cathepsin B cleavage site.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96, Zhao etal., 2008, Bioconjug Chem 19:849-59) and NK012 (Koizumi et al., 2006,Cancer Res 66:10048-56). ENZ-2208 utilizes a branched PEG to link about3.5 to 4 molecules of SN-38 per PEG, whereas NK012 is a micellenanoparticle containing 20% SN-38 by weight. With our ADC, thisdisparity (i.e., ratio of potency with free vs. conjugated SN-38)decreased as the Trop-2 expression levels increased in the tumor cells,suggesting an advantage to targeted delivery of the drug. In terms of invitro serum stability, both the CL2- and CL2A-SN-38 forms of hRS7-SN-38yielded a t/_(1/2) of ˜20 hours, which is in contrast to the shortt/_(1/2) 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56).

Treatment of tumor-bearing mice with hRS7-SN-38 (either with CL2-SN-38or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumormodels. In 4 of them, tumor regressions were observed, and in the caseof Calu-3, all mice receiving the highest dose of hRS7-SN-38 weretumor-free at the conclusion of study. Unlike in humans, irinotecan isvery efficiently converted to SN-38 by a plasma esterase in mice, with agreater than 50% conversion rate, and yielding higher efficacy in micethan in humans (Morton et al., 2000, Cancer Res 60:4206-10; Furman etal., 1999, J Clin Oncol 17:1815-24). When irinotecan was administered at10-fold higher or equivalent SN-38 levels, hRS7-SN-38 was significantlybetter in controlling tumor growth. Only when irinotecan wasadministered at its MTD of 24 mg/kg q2d×5 (37.5-fold more SN-38) did itequal the effectiveness of hRS7-SN-38. In patients, we would expect thisadvantage to favor hRS7-CL2A-SN-38 even more, because the bioconversionof irinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues (Jain, 1994, Sci Am 271:58-61). With our conjugate, 50%of the SN-38 will be released in ˜13 hours when the pH is lowered to alevel mimicking lysosomal levels (e.g., pH 5.3 at 37° C.; data notshown), whereas at the neutral pH of serum, the release rate is reducednearly 2-fold. If an irrelevant conjugate enters an acidic tumormicroenvironment, it is expected to release some SN-38 locally. Otherfactors, such as tumor physiology and innate sensitivities to the drug,will also play a role in defining this “baseline” activity. However, aspecific conjugate with a longer residence time should have enhancedpotency over this baseline response as long as there is ample antigen tocapture the specific antibody. Biodistribution studies in the SK-MES-1model also showed that if tumor antigen becomes saturated as aconsequence of successive dosing, tumor uptake of the specific conjugateis reduced, which yields therapeutic results similar to that found withan irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5 (Sapra et al., 2008, Clin Cancer Res 14:1888-96; Pastorini etal., 2010, Clin Cancer Res 16:4809-21), and preclinical studies withNK012 involved its MTD of 30 mg/kg×3 (Koizumi et al., 2006, Cancer Res66:10048-56). Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because Trop-2 recognized by hRS7 is not expressed in mice, it wasimportant to perform toxicity studies in monkeys that have a similartissue expression of Trop-2 as humans. Monkeys tolerated 0.96 mg/kg/dose(˜12 mg/m²) with mild and reversible toxicity, which extrapolates to ahuman dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a Phase I clinical trialof NK012, patients with solid tumors tolerated 28 mg/m² of SN-38 every 3weeks with Grade 4 neutropenia as dose-limiting toxicity (DLT; Hamaguchiet al., 2010, Clin Cancer Res 16:5058-66). Similarly, Phase I clinicaltrials with ENZ-2208 revealed dose-limiting febrile neutropenia, with arecommendation to administer 10 mg/m² every 3 weeks or 16 mg/m² ifpatients were administered G-CSF (Kurzrock et al., AACR-NCI-EORTCInternational Conference on Molecular Targets and Cancer Therapeutics;2009 Nov. 15-19; Boston, Mass.; Poster No C216; Patnaik et al.,AACR-NCI-EORTC International Conference on Molecular Targets and CancerTherapeutics; 2009 Nov. 15-19; Boston, Mass.; Poster No C221). Becausemonkeys tolerated a cumulative human equivalent dose of 22 mg/m², itappears that even though hRS7 binds to a number of normal tissues, theMTD for a single treatment of the hRS7 ADC could be similar to that ofthe other nontargeting SN-38 agents. Indeed, the specificity of theanti-Trop-2 antibody did not appear to play a role in defining the DLT,because the toxicity profile was similar to that of irinotecan. Moreimportantly, if antitumor activity can be achieved in humans as in micethat responded with human equivalent dose of just at 0.03 mg SN-38equivalents/kg/dose, then significant antitumor responses may berealized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting Trop-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 7 Anti-Trop-2 ADC Comprising hRS7 and Paclitaxel

A new antibody-drug conjugate (ADC) was made by conjugating paclitaxel(TAXOL®) to the hRS7 anti-human Trop-2 antibody (hRS7-paclitaxel). Thefinal product had a mean drug to antibody substitution ratio of 2.2.This ADC was tested in vitro using two different Trop-2-positive celllines as targets: BxPC-3 (human pancreatic adenocarcinoma) andMDA-MB-468 (human triple negative breast carcinoma). One day prior toadding the ADC, cells were harvested from tissue culture and plated into96-well plates at 2000 cells per well. The next day cells were exposedto free paclitaxel (6.1×10⁻¹¹ to 4×10⁻⁶ M) or the drug-equivalent ofhRS7-paclitaxel. For comparison, hRS7-SN-38 and free SN-38 were alsotested at a range of 3.84×10⁻¹² to 2.5×10⁻⁷ M. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492 nm) reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values).

The hRS7-paclitaxel ADC exhibited cytotoxic activity in the MDA-MB-468breast cell line (FIG. 15), with an IC₅₀-value approximately 4.5-foldhigher than hRS7-SN-38. The free paclitaxel was much more potent thanthe free SN-38 (FIG. 15). While the IC₅₀ for free SN-38 was 1.54×10⁻⁹ M,the IC₅₀ for free paclitaxel was less than 6.1×10⁻¹¹ M. Similar resultswere obtained for the BxPC-3 pancreatic cell line (FIG. 16) in which thehRS7-paclitaxel ADC had an IC₅₀-value approximately 2.8-fold higher thanthe hRS7-SN-38 ADC. These results show the efficacy of anti-Trop-2conjugated paclitaxel in vitro, with IC₅₀-values in the nanomolar range,similar to the hRS7-SN-38 ADC.

Example 8 Cell Binding Assay of Anti-Trop-2 Antibodies

Two different murine monoclonal antibodies against human Trop-2 wereobtained for ADC conjugation. The first, 162-46.2, was purified from ahybridoma (ATCC, HB-187) grown up in roller-bottles. A second antibody,MAB650, was purchased from R&D Systems (Minneapolis, Minn.). For acomparison of binding, the Trop-2 positive human gastric carcinoma,NCI-N87, was used as the target. Cells (1.5×10⁵/well) were plated into96-well plates the day before the binding assay. The following morning,a dose/response curve was generated with 162-46.2, MAB650, and murineRS7 (0.03 to 66 nM). These primary antibodies were incubated with thecells for 1.5 h at 4° C. Wells were washed and an anti-mouse-HRPsecondary antibody was added to all the wells for 1 h at 4° C. Wells arewashed again followed by the addition of a luminescence substrate.Plates were read using Envision plate reader and values are reported asrelative luminescent units.

All three antibodies had similar K_(D)-values of 0.57 nM for RS7, 0.52nM for 162-46.2 and 0.49 nM for MAB650. However, when comparing themaximum binding (B_(max)) of 162-46.2 and MAB650 to RS7 they werereduced by 25% and 50%, respectively (B_(Max) 11,250 for RS7, 8,471 for162-46.2 and 6,018 for MAB650) indicating different binding propertiesin comparison to RS7.

Example 9 Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a meandrug to antibody substitution ratio of 6.89. Cytotoxicity assays wereperformed to compare the MAB650-SN-38 and hRS7-SN-38 ADCs using twodifferent human pancreatic adenocarcinoma cell lines (BxPC-3 andCapan-1) and a human triple negative breast carcinoma cell line(MDA-MB-468) as targets.

One day prior to adding the ADCs, cells were harvested from tissueculture and plated into 96-well plates. The next day cells were exposedto hRS7-SN-38, MAB650-SN-38, and free SN-38 at a drug range of3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated MAB650 was used as a control atprotein equivalent doses as the MAB650-SN-38. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until an OD_(492 nm) of approximately 1.0 was reached for theuntreated cells. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values.

As shown in FIG. 17, hRS7-SN-38 and MAB650-SN-38 had similargrowth-inhibitory effects with IC₅₀-values in the low nM range which istypical for SN-38-ADCs in these cell lines. In the human Capan-1pancreatic adenocarcinoma cell line (FIG. 17A), the hRS7-SN-38 ADCshowed an IC₅₀ of 3.5 nM, compared to 4.1 nM for the MAB650-SN-38 ADCand 1.0 nM for free SN-38. In the human BxPC-3 pancreatic adenocarcinomacell line (FIG. 17B), the hRS7-SN-38 ADC showed an IC₅₀ of 2.6 nM,compared to 3.0 nM for the MAB650-SN-38 ADC and 1.0 nM for free SN-38.In the human NCI-N87 gastric adenocarcinoma cell line (FIG. 17C), thehRS7-SN-38 ADC showed an IC₅₀ of 3.6 nM, compared to 4.1 nM for theMAB650-SN-38 ADC and 4.3 nM for free SN-38.

In summary, in these in vitro assays, the SN-38 conjugates of twoanti-Trop-2 antibodies, hRS7 and MAB650, showed equal efficacies againstseveral tumor cell lines, which was similar to that of free SN-38.Because the targeting function of the anti-Trop-2 antibodies would be amuch more significant factor in vivo than in vitro, the data supportthat anti-Trop-2-SN-38 ADCs as a class would be highly efficacious invivo, as demonstrated in the Examples above for hRS7-SN-38.

Example 10 Cytotoxicity of Anti-Trop-2 ADC (162-46.2-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding adrug to antibody substitution ratio of 6.14. Cytotoxicity assays wereperformed to compare the 162-46.2-SN-38 and hRS7-SN-38 ADCs using twodifferent Trop-2-positive cell lines as targets, the BxPC-3 humanpancreatic adenocarcinoma and the MDA-MB-468 human triple negativebreast carcinoma.

One day prior to adding the ADC, cells were harvested from tissueculture and plated into 96-well plates at 2000 cells per well. The nextday cells were exposed to hRS7-SN-38, 162-46.2-SN-38, or free SN-38 at adrug range of 3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated 162-46.2 and hRS7were used as controls at the same protein equivalent doses as the162-46.2-SN-38 and hRS7-SN-38, respectively. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492 nm) nm reading ofapproximately 1.0.

Growth inhibition was measured as a percent of growth relative tountreated cells using Microsoft Excel and Prism software (non-linearregression to generate sigmoidal dose response curves which yieldIC₅₀-values).

As shown in FIG. 18A and FIG. 18B, the 162-46.2-SN-38 ADC had a similarIC₅₀-values when compared to hRS7-SN-38. When tested against the BxPC-3human pancreatic adenocarcinoma cell line (FIG. 18A), hRS7-SN-38 had anIC₅₀ of 5.8 nM, compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nM forfree SN-38. When tested against the MDA-MB-468 human breastadenocarcinoma cell line (FIG. 18B), hRS7-SN-38 had an IC₅₀ of 3.9 nM,compared to 6.1 nM for 162-46.2-SN-38 and 0.8 nM for free SN-38. Thefree antibodies alone showed little cytotoxicity to either Trop-2positive cancer cell line.

In summary, comparing the efficacies in vitro of three differentanti-Trop-2 antibodies conjugated to the same cytotoxic drug, all threeADCs exhibited equivalent cytotoxic effects against a variety of Trop-2positive cancer cell lines. These data support that the class ofanti-Trop-2 antibodies, incorporated into drug-conjugated ADCs, areeffective anti-cancer therapeutic agents for Trop-2 expressing solidtumors.

Example 11 Clinical Trials with IMMU-132 Anti-Trop-2 ADC Comprising hRS7Antibody Conjugated to SN-38

Summary

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132, an ADC of the internalizing,humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linkerto SN-38 (mean drug-antibody ratio=7.6). Trop-2 is a type Itransmembrane, calcium-transducing, protein expressed at high density(˜1×10⁵), frequency, and specificity by many human carcinomas, withlimited normal tissue expression. Preclinical studies in nude micebearing Capan-1 human pancreatic tumor xenografts have revealed IMMU-132is capable of delivering as much as 120-fold more SN-38 to tumor thanderived from a maximally tolerated irinotecan therapy.

The present Example reports the initial Phase I trial of 25 patients whohad failed multiple prior therapies (some including topoisomerase-I/IIinhibiting drugs), and the ongoing Phase II extension now reporting on69 patients, including in colorectal (CRC), small-cell and non-smallcell lung (SCLC, NSCLC, respectively), triple-negative breast (TNBC),pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (≧2⁺) in most archived tumors. In a 3+3 trial design,IMMU-132 was given on days 1 and 8 in repeated 21-day cycles, startingat 8 mg/kg/dose, then 12 and 18 mg/kg before dose-limiting neutropenia.To optimize cumulative treatment with minimal delays, phase II isfocusing on 8 and 10 mg/kg (n=30 and 14, respectively). In 49 patientsreporting related AE at this time, neutropenia≧G3 occurred in 28% (4%G4). Most common non-hematological toxicities initially in thesepatients have been fatigue (55%; ≧G3=9%), nausea (53%; ≧G3=0%), diarrhea(47%; ≧G3=9%), alopecia (40%), and vomiting (32%; ≧G3=2%). HomozygousUGT1A1*28/*28 was found in 6 patients, 2 of whom had more severehematological and GI toxicities. In the Phase I and the expansionphases, there are now 48 patients (excluding PDC) who are assessable byRECIST/CT for best response. Seven (15%) of the patients had a partialresponse (PR), including patients with CRC (N=1), TNBC (N=2), SCLC(N=2), NSCLC (N=1), and esophageal cancers (N=1), and another 27patients (56%) had stable disease (SD), for a total of 38 patients (79%)with disease response; 8 of 13 CT-assessable PDC patients (62%) had SD,with a median time to progression (TTP) of 12.7 wks compared to 8.0weeks in their last prior therapy. The TTP for the remaining 48 patientsis 12.6+ wks (range 6.0 to 51.4 wks). Plasma CEA and CA19-9 correlatedwith responses. No anti-hRS7 or anti-SN-38 antibodies were detecteddespite dosing over months. The conjugate cleared from the serum within3 days, consistent with in vivo animal studies where 50% of the SN-38was released daily, with >95% of the SN-38 in the serum being bound tothe IgG in a non-glucoronidated form, and at concentrations as much as100-fold higher than SN-38 reported in patients given irinotecan. Theseresults show that the hRS7-SN-38-containing ADC is therapeuticallyactive in metastatic solid cancers, with manageable diarrhea andneutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-38 in the serum was bound to the IgG. Lowconcentrations of SN-38G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient poll is provided in Table7.

TABLE 7 Clinical Trial Parameters Dosing regimen Once weekly for 2 weeksadministered every 21 days for up to 8 cycles. In the initialenrollment, the planned dose was delayed and reduced if ≧G2treatment-related toxicity; protocol was amended to dose delay andreduction only in the event of ≧G3 toxicity. Dose level cohorts 8, 12,18 mg/kg; later reduced to an intermediate dose level of 10 mg/kg.Cohort size Standard Phase I [3 + 3] design; expansion includes 15patients in select cancers. DLT G4 ANC ≧ 7 d; ≧G3 febrile neutropenia ofany duration; G4 Plt ≧ 5 d; G4 Hgb; Grade 4 N/V/D any duration/G3 N/V/Dfor >48 h; G3 infusion-related reactions; related ≧G3 non-hematologicaltoxicity. Maximum Acceptable Maximum dose where ≧2/6 patients tolerate1^(st) 21-d cycle w/o delay or Dose (MAD) reduction or ≧G3 toxicity.Patients Metastatic colorectal, pancreas, gastric, esophageal, lung(NSCLC, SCLC), triple-negative breast (TNBC), prostate, ovarian, renal,urinary bladder, head/neck, hepatocellular. Refractory/relapsed afterstandard treatment regimens for metastatic cancer. Prioririnotecan-containing therapy NOT required for enrollment. No bulkylesion >5 cm. Must be 4 weeks beyond any major surgery, and 2 weeksbeyond radiation or chemotherapy regimen. Gilbert's disease or known CNSmetastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (FIG. 19) and Time to Progression (TTP; FIG.20) were determined. To summarize the Best Response data, of 8assessable patients with TNBC (triple-negative breast cancer), therewere 2 PR (partial response), 4 SD (stable disease) and 2 PD(progressive disease) for a total response [PR+SD] of 6/8 (75%). ForSCLC (small cell lung cancer), of 4 assessable patients there were 2 PR,0 SD and 2 PD for a total response of 2/4 (50%). For CRC (colorectalcancer), of 18 assessable patients there were 1 PR, 11 SD and 6 PD for atotal response of 12/18 (67%). For esophageal cancer, of 4 assessablepatients there were 1 PR, 2 SD and 1 PD for a total response of 3/4(75%). For NSCLC (non-small cell lung cancer), of 5 assessable patientsthere were 1 PR, 3 SD and 1 PD for a total response of 4/5 (80%). Overall patients treated, of 48 assessable patients there were 7 PR, 27 SDand 14 PD for a total response of 34/48 (71%). These results demonstratethat the anti-Trop-2 ADC (hRS7-SN-38) showed significant clinicalefficacy against a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 8. As apparent from the data of Table 8, the therapeutic efficacyof hRS7-SN-38 was achieved at dosages of ADC showing an acceptably lowlevel of adverse side effects.

TABLE 8 Related Adverse Events Listing for IMMU-132-01 Criteria: Total≧10% or ≧Grade 3 N = 47 patients TOTAL Grade 3 Grade 4 Fatigue 55% 4(9%) 0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2(4%) Alopecia 40% — — Vomiting 32% 1 (2%) 0 Anemia 13% 2 (4%) 0Dysgeusia 15% 0 0 Pyrexia 13% 0 0 Abdominal pain 11% 0 0 Hypokalemia 11%1 (2%) 0 WBC Decrease  6% 1 (2%) 0 Febrile Neutropenia  6% 1 (2%) 2 (4%)Deep vein thrombosis  2% 1 (2%) 0 Grading by CTCAE v 4.0

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62 year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo 5FU. She presented with multiple lesionsprimarily in the liver (3+Trop-2 by immunohistology), entering thehRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year afterinitial diagnosis. On her first CT assessment, a PR was achieved, with a37% reduction in target lesions (not shown). The patient continuedtreatment, achieving a maximum reduction of 65% decrease after 10 monthsof treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65 year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of caboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65 year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topo-II inhibitor) that ended after 2 months with no response, followedwith topotecan (Topo-I inhibitor) that ended after 2 months, also withno response, she received local XRT (3000 cGy) that ended 1 month later.However, by the following month progression had continued. The patientstarted with IMMU-132 the next month (12 mg/kg; reduced to 6.8 mg/kg;Trop-2 expression 3+), and after two months of IMMU-132, a 38% reductionin target lesions, including a substantial reduction in the main lunglesion occurred (not shown). The patient progressed 3 months later afterreceiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies.

In conclusion, at the dosages used, the primary toxicity was amanageable neutropenia, with few Grade 3 toxicities. IMMU-132 showedevidence of activity (PR and durable SD) in relapsed/refractory patientswith triple-negative breast cancer, small cell lung cancer, non-smallcell lung cancer, colorectal cancer and esophageal cancer, includingpatients with a previous history of relapsing on topoisomerase-Iinhibitor therapy. These results show efficacy of the anti-Trop-2 ADC ina wide range of cancers that are resistant to existing therapies.

Example 12 Treatment of Triple Negative Breast Cancer withPro-2-PDox-hRS7 ADC

pro-2-PDox-hRS7 ADC is prepared as described in the Examples above.Patients with triple-negative breast cancer who had failed at least twostandard therapies receive 3 cycles of 70 mg pro-2-PDox-hRS7 injectedi.v. every 3 weeks. Objective responses are observed at this dose levelof pro-2-PDox-hRS7, with an average decrease in tumor volume of 35%,after two cycles of therapy. All serum samples evaluated for humananti-hRS7 antibody (HAHA) are negative.

Example 13 Treatment of Metastatic Colon Cancer with Pro-2-PDox-hRS7 ADC

A 52-year old man with metastatic colon cancer (3-5 cm diameters) to hisleft and right liver lobes, as well as a 5 cm metastasis to his rightlung, and an elevated blood CEA value of 130 ng/mL, is treated with a150 mg dose of hRS7 anti-Trop-2 conjugated with pro-2-PDox at 4 drugmolecules per IgG, administered by slow intravenous infusion every otherweek for 4 doses. Upon CT evaluation 8 weeks from treatment begin, a 25%reduction of the total mean diameters of the 3 target lesions ismeasured, thus constituting a good stable disease response by RECIST1.1criteria. Repeated courses of therapy continue as his neutropenianormalizes.

Example 14 Treatment of Metastatic Pancreatic Cancer withPro-2-PDox-hRS7 ADC

A 62-year old man with metastatic ductal adenocarcinoma of the pancreas,who has relapsed after prior therapies with FOLFIRINOX followed byNab-taxol (Abraxane®) plus gemcitabine is given hRS7-pro-2-PDox ADC at adose of 120 mg every other week for 4 courses, and after a 3-week delay,another course of 2 injections 2 weeks apart are given intravenously.The patient shows some nausea and transient diarrhea with the therapy,and also Grade 3 neutropenia after the first course, which recoversbefore the second course of therapy. CT measurements made at 8 weeksfollowing start of therapy show an 18% shrinkage of the sum of the 3target lesions in the liver, as compared to the pretreatment baselinemeasurements, constituting stable disease by RECIST 1.1 criteria. Also,the patient's CA19-9 blood titer is reduced by 55% from a baseline valueof 12,400. His general symptoms of weakness, fatigue and abdominaldiscomfort also improve considerably, including regaining his appetiteand a weight increase of 2 kg during the following 6 weeks.

Example 15 Combining Antibody-Targeted Radiation (Radioimmunotherapy)and Anti-Trop-2-SN-38 ADC Improves Pancreatic Cancer Therapy

We previously reported effective anti-tumor activity in nude micebearing human pancreatic tumors with ⁹⁰Y-humanized PAM4 IgG (hPAM4;⁹⁰Y-clivatuzumab tetraxetan) that was enhanced when combined withgemcitabine (GEM) (Gold et al., Int J. Cancer 109:618-26, 2004; ClinCancer Res 9:3929S-37S, 2003). These studies led to clinical testing offractionated ⁹⁰Y-hPAM4 IgG combined with GEM that is showing encouragingobjective responses. While GEM is known for its radiosensitizingability, alone it is not a very effective therapeutic agent forpancreatic cancer and its dose is limited by hematologic toxicity, whichis also limiting for ⁹⁰Y-hPAM4 IgG.

As discussed in the Examples above, an anti-Trop-2 ADC composed of hRS7IgG linked to SN-38 shows anti-tumor activity in various solid tumors.This ADC is very well tolerated in mice (e.g., ≧60 mg), yet just 4.0 mg(0.5 mg, twice-weekly×4) is significantly therapeutic. Trop-2 is alsoexpressed in most pancreatic cancers.

The present study examined combinations of ⁹⁰Y-hPAM4 IgG with RS7-SN-38in nude mice bearing 0.35 cm³ subcutaneous xenografts of the humanpancreatic cancer cell line, Capan-1. Mice (n=10) were treated with asingle dose of ⁹⁰Y-hPAM4 IgG alone (130 μCi, i.e., the maximum tolerateddose (MTD) or 75 Ci), with RS7-SN-38 alone (as above), or combinationsof the 2 agents at the two ⁹⁰Y-hPAM4 dose levels, with the first ADCinjection given the same day as the ⁹⁰Y-hPAM4. All treatments weretolerated, with ≦15% loss in body weight. Objective responses occurredin most animals, but they were more robust in both of the combinationgroups as compared to each agent given alone. All animals in the0.13-mCi ⁹⁰Y-hPAM4 IgG+ hRS7-SN-38 group achieved a tumor-free statewithin 4 weeks, while other animals continued to have evidence ofpersistent disease. These studies provide the first evidence thatcombined radioimmunotherapy and ADC enhances efficacy at safe doses.

In the ongoing PAM4 clinical trials, a four week clinical treatmentcycle is performed. In week 1, subjects are administered a dose of¹¹¹In-hPAM4, followed at least 2 days later by gemcitabine dose. Inweeks 2, 3 and 4, subjects are administered a ⁹⁰Y-hPAM4 dose, followedat least 2 days later by gemcitabine (200 mg/m²). Escalation started at3×6.5 mCi/m². The maximum tolerated dose in front-line pancreatic cancerpatients was 3×15 mCi/m² (hematologic toxicity is dose-limiting). Of 22CT-assessable patients, the disease control rate (CR+PR+SD) was 68%,with 5 (23%) partial responses and 10 (45%) having stabilization as bestresponse by RECIST criteria.

Preparation of Antibody-Drug Conjugate (ADC)

The SN-38 conjugated hRS7 antibody was prepared as described above andaccording to previously described protocols (Moon et al. J Med Chem2008, 51:6916-6926; Govindan et al., Clin Cancer Res 2009.15:6052-6061). A reactive bifunctional derivative of SN-38 (CL2A-SN-38)was prepared. The formula of CL2A-SN-38 is(maleimido-[x]-Lys-PABOCO-20-O-SN-38, where PAB is p-aminobenzyl and ‘x’contains a short PEG). Following reduction of disulfide bonds in theantibody with TCEP, the CL2A-SN-38 was reacted with reduced antibody togenerate the SN-38 conjugated RS7.

⁹⁰Y-hPAM4 is prepared as previously described (Gold et al., Clin CancerRes 2003, 9:3929S-37S; Gold et al., Int J Cancer 2004, 109; 618-26).

Combination RAIT+ADC

The Trop-2 antigen is expressed in most epithelial cancers (lung,breast, prostate, ovarian, colorectal, pancreatic) and hRS7-SN-38conjugates are being examined in various human cancer-mouse xenograftmodels. Initial clinical trials with ⁹⁰Y-hPAM4 IgG plus radiosensitizingamounts of GEM are encouraging, with evidence of tumor shrinkage orstable disease. However, therapy of pancreatic cancer is verychallenging. Therefore, a combination therapy was examined to determinewhether it would induce a better response. Specifically, administrationof hRS7-SN-38 at effective, yet non-toxic doses was combined with RAITwith ⁹⁰Y-hPAM4 IgG.

The results demonstrated that the combination of hRS7-SN-38 with⁹⁰Y-hPAM4 was more effective than either treatment alone, or the sum ofthe individual treatments (not shown). At a dosage of 75 μCi ⁹⁰Y-hPAM4,only 1 of 10 mice was tumor-free after 20 weeks of therapy (not shown),the same as observed with hRS7-SN-38 alone (not shown). However, thecombination of hRS7-SN-38 with ⁹⁰Y-hPAM4 resulted in 4 of 10 mice thatwere tumor-free after 20 weeks (not shown), and the remaining subjectsshowed substantial decrease in tumor volume compared with eithertreatment alone (not shown). At 130 μCi ⁹⁰Y-hPAM4 the difference waseven more striking, with 9 of 10 animals tumor-free in the combinedtherapy group compared to 5 of 10 in the RAIT alone group (not shown).These data demonstrate the synergistic effect of the combination ofhRS7-SN-38 with ⁹⁰Y-hPAM4. RAIT+ADC significantly improved time toprogression and increased the frequency of tumor-free treatment. Thecombination of ADC with hRS7-SN-38 added to the MTD of RAIT with⁹⁰Y-hPAM4 had minimal additional toxicity, indicated by the % weightloss of the animal in response to treatment (not shown).

The effect of different sequential treatments on tumor survivalindicated that the optimal effect is obtained when RAIT is administeredfirst, followed by ADC (not shown). In contrast, when ADC isadministered first followed by RAIT, there is a decrease in theincidence of tumor-free animals (not shown). Neither unconjugated hPAM4nor hRS7 antibodies had anti-tumor activity when given alone (notshown).

Example 16 Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM5 humanized MAb, hMN-14 (also known as labetuzumab), theanti-CD22 humanized MAb, hLL2 (also known as epratuzumab), the anti-CD20humanized MAb, hA20 (also known as veltuzumab), the anti-EGP-1 humanizedMAb, hRS7, and anti-mucin humanized MAb, hPAM4 (also known asclivatuzumab), were conjugated to SN-38 using a CL2A linker. Eachantibody was reduced with dithiothreitol (DTT), used in a 50-to-70-foldmolar excess, in 40 mM PBS, pH 7.4, containing 5.4 mM EDTA, at 37° C.(bath) for 45 min. The reduced product was purified by size-exclusionchromatography and/or diafiltration, and was buffer-exchanged into asuitable buffer at pH 6.5. The thiol content was determined by Ellman'sassay, and was in the 6.5-to-8.5 SH/IgG range. Alternatively, theantibodies were reduced with Tris (2-carboxyethyl) phosphine (TCEP) inphosphate buffer at pH in the range of 5-7, followed by in situconjugation. The reduced MAb was reacted with ˜10-to-15-fold molarexcess of CL2A-SN-38 using DMSO at 7-15% v/v as co-solvent, andincubating for 20 min at ambient temperature. The conjugate was purifiedby centrifuged SEC, passage through a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. This way, theSN-38/MAb substitution ratios were determined. The purified conjugateswere stored as lyophilized formulations in glass vials, capped undervacuum and stored in a −20° C. freezer. SN-38 molar substitution ratios(MSR) obtained for these conjugates were typically in the 5-to-7 range

Example 17 Therapy of Advanced Colon Cancer Patient Refractory to PriorChemo-Immunotherapy, Using Only IMMU-130 (Labetuzumab-SN-38)

The patient is a 50-year-old man with a history of stage-IV metastaticcolonic cancer, first diagnosed in 2008 and given a colectomy andpartial hepatectomy for the primary and metastatic colonic cancers,respectively. He then received chemotherapy, as indicated in FIG. 8,which included irinotecan, oxaliplatin, FOLFIRINOX (5-fluoruracil,leucovorin, irinotecan, oxaliplatin), and bevacizumab, as well asbevacizumab combined with 5-fluorouracil/leucovorin, for almost 2 years.Thereafter, he was given courses of cetuximab, either alone or combinedwith FOLFIRI (leucovorin, 5-flurouracil, irinotecan) chemotherapy duringthe next year or more. In 2009, he received radiofrequency ablationtherapy to his liver metastasis while under chemo-immunotherapy, and inlate 2010 he underwent a wedge resection of his lung metastases, whichwas repeated a few months later, in early 2011. Despite havingchemo-immunotherapy in 2011, new lung metastases appeared at the end of2011, and in 2012, both lung and liver metastases were visualized. Hisbaseline plasma carcinoembryonic antigen (CEA) titer was 12.5 ng/mL just. . . before undergoing the antibody-drug therapy with IMMU-130. Theindex lesions chosen by the radiologist for measuring tumor size changeby computed tomography were the mid-lobe of the right lung and the livermetastases, both totaling 91 mm as the sum of their longest diameters atthe baseline prior to IMMU-130 (anti-CEACAM5-SN-38) therapy.

This patient received doses of 16 mg/kg of IMMU-130 by slow IV infusionevery other week for a total of 17 treatment doses. The patienttolerated the therapy well, having only a grade 1 nausea, diarrhea andfatigue after the first treatment, which occurred after treatments 4 and5, but not thereafter, because he received medication for theseside-effects. After treatment 3, he did show alopecia (grade 2), whichwas present during the subsequent therapy. The nausea, diarrhea, andoccasional vomiting lasted only 2-3 days, and his fatigue after thefirst infusion lasted 2 weeks. Otherwise, the patient tolerated thetherapy well. Because of the long duration of receiving this humanized(CDR-grafted) antibody conjugated with SN-38, his blood was measured foranti-labetuzumab antibody, and none was detected, even after 16 doses.

The first computed tomography (CT) measurements were made after 4treatments, and showed a 28.6% change from the sum of the measurementsmade at baseline, prior to this therapy, in the index lesions. After 8treatments, this reduction became 40.6%, thus constituting a partialremission according to RECIST criteria. This response was maintained foranother 2 months, when his CT measurements indicated that the indexlesions were 31.9% less than the baseline measurements, but somewhathigher than the previous decrease of 40.6% measured. Thus, based oncareful CT measurements of the index lesions in the lung and liver, thispatient, who had failed prior chemotherapy and immunotherapy, includingirinotecan (parent molecule of SN-38), showed an objective response tothe active metabolite of irintotecan (or camptotechin), SN-38, whentargeted via the anti-CEACAM5 humanized antibody, labetuzumab (hMN-14).It was surprising that although irinotecan (CPT-11) acts by releasingSN-38 in vivo, the SN-38 conjugated anti-CEACAM5 antibody provedeffective in a colorectal cancer patient by inducing a partial responseafter the patient earlier failed to respond to his lastirinotecan-containing therapy. The patient's plasma CEA titer reductionalso corroborated the CT findings: it fell from the baseline level of12.6 ng/mL to 2.1 ng/mL after the third therapy dose, and was between1.7 and 3.6 ng/mL between doses 8 and 12. The normal plasma titer of CEAis usually considered to be between 2.5 and 5.0 ng/mL, so this therapyeffected a normalization of his CEA titer in the blood.

Example 18 Therapy of a Patient with Advanced Colonic Cancer withIMMU-130

This patient is a 75-year-old woman initially diagnosed with metastaticcolonic cancer (Stage IV). She has a right partial hemicolectomy andresection of her small intestine and then receives FOLFOX,FOLFOX+bevacizumab, FOLFIRI+ramucirumab, and FOLFIRI+cetuximab therapiesfor a year and a half, when she shows progression of disease, withspread of disease to the posterior cul-de-sac, omentum, with ascites inher pelvis and a pleural effusion on the right side of her chest cavity.Her baseline CEA titer just before this therapy is 15 ng/mL. She isgiven 6 mg/kg IMMU-130 (anti-CEACAM5-SN-38) twice weekly for 2consecutive weeks, and then one week rest (3-week cycle), for more than20 doses, which is tolerated very well, without any major hematologicalor non-hematological toxicities. Within 2 months of therapy, her plasmaCEA titer shrinks modestly to 1.3 ng/mL, but at the 8-week evaluationshe shows a 21% shrinkage of the index tumor lesions, which increases toa 27% shrinkage at 13 weeks. Surprisingly, the patient's ascites andpleural effusion both decrease (with the latter disappearing) at thistime, thus improving the patient's overall status remarkably. Thepatient continues her investigational therapy.

Example 19 Gastric Cancer Patient with Stage IV Metastatic DiseaseTreated with IMMU-130

The patient is a 52-year-old male who sought medical attention becauseof gastric discomfort and pain related to eating for about 6 years, andwith weight loss during the past 12 months. Palpation of the stomacharea reveals a firm lump which is then gastroscoped, revealing anulcerous mass at the lower part of his stomach. This is biopsied anddiagnosed as a gastric adenocarcinoma. Laboratory testing reveals nospecific abnormal changes, except that liver function tests, LDH, andCEA are elevated, the latter being 10.2 ng/mL. The patent then undergoesa total-body PET scan, which discloses, in addition to the gastrictumor, metastatic disease in the left axilla and in the right lobe ofthe liver (2 small metastases). The patient has his gastric tumorresected, and then has baseline CT measurements of his metastatictumors. Four weeks after surgery, he receives 3 courses of combinationchemotherapy consisting of a regimen of cisplatin and 5-fluorouracil(CF), but does not tolerate this well, so is switched to treatment withdocetaxel. It appears that the disease is stabilized for about 4 months,based on CT scans, but then the patient's complaints of further weightloss, abdominal pain, loss of appetite, and extreme fatigue causerepeated CT studies, which show increase in size of the metastases by asum of 20% and a suspicious lesion at the site of the original gastricresection.

The patient is then given experimental therapy with IMMU-130(anti-CEACAM5-SN-38) on a weekly schedule of 8 mg/kg. He tolerates thiswell, but after 3 weeks shows a grade 2 neutropenia and grade 1diarrhea. His fourth infusion is postponed by one week, and then theweekly infusions are reinstituted, with no evidence of diarrhea orneutropenia for the next 4 injection. The patient then undergoes a CTstudy to measure his metastatic tumor sizes and to view the originalarea of gastric resection. The radiologist measures, according to RECISTcriteria, a decrease of the sum of the metastatic lesions, compared tobaseline prior to IMMU-130 therapy, of 23%. There does not seem to beany clear lesion in the area of the original gastric resection. Thepatient's CEA titer at this time is 7.2 ng/mL, which is much reducedfrom the pre-IMMU-130 baseline value of 14.5 ng/mL. The patientcontinues on weekly IMMU-130 therapy at the same dose of 8.0 mg/kg, andafter a total of 13 infusions, his CT studies show that one livermetastasis has disappeared and the sum of all metastatic lesions isdecreased by 41%, constituting a partial response by RECIST. Thepatient's general condition improves and he resumes his usual activitieswhile continuing to receive a maintenance therapy of 8 mg/kg IMMU-130every third week for another 4 injections. At the last measurement ofblood CEA, the value is 4.8 ng/mL, which is within the normal range fora smoker, which is the case for this patient.

Example 20 Therapy of Advanced Metastatic Colon Cancer with Anti-CEACAM5Immunoconjugate

The patient is a 50-year-old male who fails prior therapies formetastatic colon cancer. The first line of therapy isFOLFIRINOX+AVASTIN® (built up in a stepwise manner) starting with IROX(Irinotecan+Oxaliplatin) in the first cycle. After initiating thistreatment the patient has a CT that shows decrease in the size of livermetastases. This is followed by surgery to remove tumor tissue. Adjuvantchemotherapy is a continuation of the first line regimen (without theIROX part) that resulted in a transient recurrence-free period. Afterabout a 1 year interval, a CT reveals the recurrence of livermetastases. This leads to the initiation of the second line regimen(FOLFIRI+Cetuximab). Another CT shows a response in liver metastases.Then RF ablation of liver metastases is performed, followed bycontinuation of adjuvant chemotherapy with FOLFIRINOX+Cetuximab,followed by maintenance Cetuximab for approximately one year. Another CTscan shows no evidence of disease. A further scan shows possible lungnodules, which is confirmed. This leads to a wedge resection of the lungnodules. Subsequently FOLFIRI+Cetuximab is restarted and continued.Later CT scans show both lung and liver metastases.

At the time of administration of the hMN-14-SN-38 immunoconjugate, thepatient has advanced metastatic colon cancer, with metastases of bothlung and liver, which is unresponsive to irinotecan (camptothecin). ThehMN-14-SN-38 immunoconjugate is administered at a dosage of 12 mg/kg,which is repeated every other week. The patient shows a partial responsewith reduction of metastatic tumors by RECIST criteria.

Of note is that only one patient in this 12 mg/kg (given every otherweek) cohort shows a grade 2 hematological (neutropenia) and mostpatients have grade 1 or 2 nausea, vomiting, or alopecia—which are signsof activity of the antibody-drug conjugate, but well tolerated. Theeffect of the antibody moiety in improved targeting of the camptothecinaccounts for the efficacy of the SN-38 moiety in the cancer that hadbeen previously resistant to unconjugated irinotecan.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

What is claimed is:
 1. A method of treating cancer comprisingadministering to a human patient with a Trop-2 positive cancer animmunoconjugate comprising SN-38 conjugated to an anti-Trop-2 antibodyor antigen-binding fragment thereof, wherein the immunoconjugate isadministered at a dosage of between 4 mg/kg and 16 mg/kg.
 2. The methodof claim 1, wherein the dosage is selected from the group consisting of4 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12 mg/kg, and 16mg/kg.
 3. The method of claim 1, wherein the dosage is between 8 mg/kgto 10 mg/kg.
 4. The method of claim 1, wherein the antibody is hRS7(anti-Trop-2).
 5. The method of claim 1, wherein the cancer is agastrointestinal cancer.
 6. The method of claim 5, wherein the cancer iscolorectal cancer, esophageal cancer or gastric cancer.
 7. The method ofclaim 1, wherein the cancer is colorectal, lung, stomach, urinarybladder, renal, breast, ovarian, uterine or prostatic cancer
 8. Themethod of claim 7, wherein the breast cancer is triple negative breastcancer (TNBC).
 9. The method of claim 1, wherein the cancer ismetastatic.
 10. The method of claim 1, wherein the cancer is a solidtumor and the treatment results in a reduction in tumor size of at least15%, at least 20%, at least 30%, or at least 40%.
 11. The method ofclaim 8, further comprising reducing in size or eliminating themetastases.
 12. The method of claim 1, wherein the cancer is refractoryto other therapies but responds to the immunoconjugate.
 13. The methodof claim 1, wherein there is a CL2A linker between the SN-38 and theantibody and the structure of the immunoconjugate is MAb-CL2A-SN-38


14. The method of claim 1, wherein there are 6 or more SN-38 moleculesattached to each antibody molecule.
 15. The method of claim 1, whereinthere are 6-8 SN-38 molecules attached to each antibody molecule. 16.The method of claim 1, wherein there are 7-8 SN-38 molecules attached toeach antibody molecule.
 17. The method of claim 1, wherein the antibodyis an IgG1 or IgG4 antibody.
 18. The method of claim 1, wherein theantibody has an allotype selected from the group consisting of G1m3,G1m3,1, G1m3,2, G1m3,1,2, nG1m1, nG1m1, 2 and Km3 allotypes.
 19. Themethod of claim 1, wherein the immunoconjugate dosage is administered tothe human subject once or twice a week on a schedule with a cycleselected from the group consisting of: (i) weekly; (ii) every otherweek; (iii) one week of therapy followed by two, three or four weeksoff; (iv) two weeks of therapy followed by one, two, three or four weeksoff; (v) three weeks of therapy followed by one, two, three, four orfive weeks off; (vi) four weeks of therapy followed by one, two, three,four or five weeks off; (vii) five weeks of therapy followed by one,two, three, four or five weeks off; and (viii) monthly.
 20. The methodof claim 19, wherein the cycle is repeated 4, 6, 8, 10, 12, 16 or 20times.
 21. The method of claim 1, wherein the immunoconjugate isadministered in combination with one or more therapeutic modalitiesselected from the group consisting of unconjugated antibodies,radiolabeled antibodies, drug-conjugated antibodies, toxin-conjugatedantibodies, gene therapy, chemotherapy, therapeutic peptides, cytokinetherapy, oligonucleotides, localized radiation therapy, surgery andinterference RNA therapy.
 22. The method of claim 21, wherein theimmunoconjugate is administered simultaneously with the one or moretherapeutic modalities.
 23. The method of claim 22, wherein theimmunoconjugate is administered separately from the one or moretherapeutic modalities.
 24. The method of claim 21, wherein theunconjugated antibody is selected from the group consisting oflambrolizumab, nivolumab and ipilimumab.
 25. The method of claim 21,wherein the drug, toxin or chemotherapeutic agent is selected from thegroup consisting of 5-fluorouracil, afatinib, aplidin, azaribine,anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine,bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,crizotinib, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel,dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine(2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin,erlotinib, entinostat, estrogen receptor binding agents, etoposide(VP16), etoposide glucuronide, etoposide phosphate, exemestane,fingolimod, flavopiridol, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferaseinhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide,imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 26. The method ofclaim 1, wherein the cancer is metastatic colon cancer and the patienthas failed FOLFIRI or FOLFOX chemotherapy prior to administration of theimmunoconjugate.